Shop All Labels & Labeling Kits

EZ-Link™ Maleimide-PEG2-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link Maleimide-PEG2-Biotin is a mid-length, maleimide-activated, sulfhydryl-reactive biotinylation reagent that contains a 2-unit ethylene glycol in its spacer arm for increased water-solubility characteristics.

Features of EZ-Link Maleimide-PEG2-Biotin:

Protein labeling—biotinylate antibodies or other proteins for use in protein methods
Thiol-reactive—reacts with sulfhydryls (-SH), such as the side-chain of cysteine (C)
Maleimide-activated—perform reactions at pH 6.5 to 7.5 in buffers such as PBS
Pegylated—spacer arm contains a hydrophilic, 2-unit, polyethylene glycol (PEG) group
Enhances solubility—pegylation imparts water solubility to the biotinylated molecule, helping to prevent aggregation of biotinylated antibodies stored in solution
Irreversible—forms permanent thioether bonds; spacer arm cannot be cleaved
Solubility—can be dissolved directly in aqueous buffers for labeling reactions
Medium length—spacer arm (total length added to target) is 29.1 angstroms

Maleimide-PEG2-Biotin enables simple and efficient biotinylation of antibodies, cysteine-containing peptides and other thiol-containing molecules. The maleimide group reacts specifically and efficiently with reduced thiols (sulfhydryl groups,—SH) at pH 6.5 to 7.5 to form stable thioether bonds. The hydrophilic, 2-unit polyethylene glycol (PEG) spacer arm imparts water solubility that is transferred to the biotinylated molecule, thus reducing aggregation of labeled proteins stored in solution. The PEG segment adds length and flexibility to the spacer arm, minimizing steric hindrance involved with binding to avidin molecules.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.

Biotinylation reagents differ in reactivity, length, solubility, cell permeability and cleavability. Three types of sulfhydryl-reactive compounds are available: maleimido, iodoacetyl and pyridyldithiol. Maleimide reagents specifically react with sulfhydryl groups (-SH) in near-neutral buffers to form permanent thioether bonds.

In proteins, sulfhydryls exist where there are cysteine (C) residues. Cystine disulfide bonds must be reduced to make sulfhydryl groups available for labeling. Hinge-region disulfide bridges of antibodies can be selectively reduced to make functional half-antibodies that can be labeled.

Alexa Fluor™ 594 Hydrazide, for microinjection (Invitrogen™)

Alexa Fluor® 594 Hydrazide is useful as a cell tracer and as a reactive dye for labeling aldehydes or ketones in polysaccharides or glycoproteins. This version is formatted as a ready-to-use solution that is dissolved in a 200 mM KCl solution and filter sterilized.

Alexa Fluor® 594 is a bright, red fluorescent dye. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 594 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 594 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

Detailed information about this AlexaFluor® hydrazide:

• Fluorophore label : Alexa Fluor® 594 dye
• Reactive group: hydrazide
• Reactivity: Aldehydes or keytones in polysaccharides or glycoproteins
• Ex/Em of the conjugate: 588/613 nm
• Extinction coefficient: 97,000 cm-1M-1
• Spectrally similar dyes: Texas Red
• Molecular weight: 758.79

Cell Tracking and Tracing Applications
Alexa Fluor® hydrazides and hydroxlamines are useful as low molecular weight, membrane-impermeant, aldehyde-fixable cell tracers, exhibiting brighter fluorescence and greater photostability than cell tracers derived from other spectrally similar fluorophores. They are easily loaded into cells by microinjection, infusion from patch pipette, or uptake induced by our Influx™ Pinocytic Cell-Loading Reagent. Learn more about cell tracking and tracing.

Glycoprotein and Polysaccharide Labeling Applications
The Alexa Fluor® hydrazides and hydroxlamines are reactive molecules that can be used to add a fluorescent label to biomolecules containing aldehydes or ketones. Aldehydes and ketones can be introduced into polysaccharides and glycoproteins by periodate-mediated oxidation of vicinal diols. Galactose oxidase can also be used to oxidize terminal galactose residues of glycoproteins to aldehydes.

Hydrazide vs Hydroxylamine
Hydrazine derivatives react with ketones and aldehydes to yield relatively stable hydrazones. Hydroxylamine derivatives (aminooxy compounds) react with aldehydes and ketones to yield oximes. Oximes are superior to hydrazones with respect to hydrolytic stability. Both hydrazones and oximes can be reduced with sodium borohydride (NaBH4) to further increase the stability of the linkage.

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

Related Products
DMSO (dimethylsulfoxide) (D12345)
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

DyLight™ 650 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 650 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 650 used to fluorescently label sulfhydryl-containing peptides, proteins, and other biomolecular probes.

DyLight 650 provides vibrant far-red fluorescence with comparable or improved performance over other dyes, including Alexa Fluor™ 647 and Cy5™ dye, over a broad pH range (pH 4-9). The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates.

Features of DyLight 650 Maleimide:

High performance—DyLight 650 fluoresces brighter than Alexa Fluor 647 and Cy5
Specific—maleimide-activated dye labels proteins and other molecules at reduced sulfhydryls (-SH)
Efficient labeling methods—well-characterized chemistry and optimized protocols provide for reliable, high-quality labeling
Optimized antibody labeling procedure—complete protocol for IgG reduction and labeling and calculating the labeling efficiency

Applications:
• Antibody labeling for immunofluorescence applications, including immunocytochemistry (ICC), immunohistochemistry (IHC), Western blotting and ELISA assay
• Target macromolecule labeling for in vitro and in vivo fluorescent detection strategies

DyLight 650 Sulfhydryl-Reactive Dye is activated with a maleic acid imide (maleimide) moiety to form a reactive alkylation reagent. Labeling occurs through reaction of the maleimide-activated dye with reduced sulfhydryl groups (-SH) to form stable thioether bonds. Maleimides are specific for sulfhydryl groups between pH 6.5–7.5. Learn more about maleimide chemistry.

SiteClick™ Qdot™ 525 Antibody Labeling Kit (Invitrogen™)

Create a perfectly labeled antibody with the SiteClick™ Qdot® 525 Antibody Labeling Kit. This kit replaces the conventional Qdot® 525 Antibody Conjugation Kit (Q22041MP). Unlike the conventional amine-thiol crosslinker method, SiteClick™ labeling specifically attaches the label to the heavy chains of an IgG antibody ensuring that the antigen binding domains remain available for binding to your antigen target. This site selectivity is achieved by targeting the carbohydrate domains present on essentially all IgG antibodies regardless of isotype and host species. In addition, no harsh reduction steps are required, and the labeling is consistent and reproducible each time it is performed. Depending upon the label, the resulting SiteClick™ labeled antibody can be used in flow cytometry, fluorescence imaging, or western blot detection.

Important Features of the SiteClick™ Qdot® 525 Antibody Labeling Kit:

• Contains everything required to label 100 µg of IgG antibody
• Easy to follow step-by-step protocol
• Highly efficient, site-specific, reproducible labeling chemistry results in high quality antibody conjugate.
• Qdot® 525 labels can be used in confocal or traditional fluoresce microscopy.
• In flow cytometry, Qdot® 525 can be excited by the 488 nm line of the argon-ion laser, or alternatively via excitation at 405 nm. This is true of all Qdot® fluorophores.

Qdot® Fluorophores Are Our Brightest Labels
Antibody conjugates made with Qdot® fluorophores produce fluorescence output that surpasses that of traditional organic dyes. Paired with the correct optical filters, Qdot® nanocrystals are as much as 50 times brighter. Read more about Qdot® nanocrystals or review additional product details in Qdot® Nanocrystals—Section 6.6 in the Molecular Probes® Handbook.

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits (see Antibody Labeling from A to Z). To learn more about our various kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in the Molecular Probes® Handbook.

Custom SiteClick™ Antibody Labeling Services
If you have an antibody that is considered "difficult to label" or has lost activity after labeling using a conventional method, please contact our custom service representatives to determine whether the SiteClick™ Antibody Labeling Service would be right for your antibody. We offer complete custom SiteClick™ antibody labeling services with the option of multiple detection molecules including biotin, Alexa Fluor® dyes, Qdot® fluorophores, R-PE, and others.

Qdot™ 800 ITK™ Amino (PEG) Quantum Dots (Invitrogen™)

Qdot® 800 ITK™ amino (PEG) quantum dots are the ideal starting material for preparing custom conjugates of ultrabright and photostable fluorescently labeled proteins or other biopolymers. These probes are functionalized with amine-derivatized PEG, which prevents non-specific interactions and provides a convenient handle for conjugation. The amino quantum dots react efficiently with isothiocyanates and succinimidyl esters, or with native carboxylic acids using water-soluble carbodiimides such as EDC. Such derivatives may be used for various labeling and tracking applications that require ultrabright and stable fluorescence. Our Qdot® ITK™ amino quantum dots are provided as 8 µM solutions and are available in 8 colors of Qdot® probes.

Important Features of Qdot® ITK™ Amino Quantum Dots:
• Qdot® 800 ITK™ amino quantum dot has emission maxima of ~800 nm
• Extremely photostable and bright fluorescence
• Efficiently excited with single-line excitation sources
• Narrow emission, large stokes shift
• Available in multiple colors
• Ideal for various labeling and tracking applications


Properties of Qdot® Nanocrystals
Qdot® probes are ideal for imaging and labeling applications that require bright fluorescent signals and/or real-time tracking. Unique among fluorescent reagents, all nine available colors of Qdot® probes can be simultaneously excited with a single (UV to blue-green) light source. This property makes these reagents excellent for economical and user-friendly multiplexing applications. Qdot® labels are based on semiconductor nanotechnology and are similar in scale to moderately sized proteins.

About the Innovator’s Tool Kit Qdot® ITK™ Reagents
These Qdot® ITK™ probes are ideal for researchers who wish to prepare specific (non-stocked) conjugates for their applications and need customizable conjugation functionality.

Other Forms of Qdot® Nanocrystals are Available
In addition to the amine-derivatized form, we offer Qdot® ITK™ quantum dots with carboxyl and aliphatic hydrocarbon modifications. We’ve also developed a wide range of Qdot® nanocrystals conjugates and labeling kits. Investigate the properties of Qdot® nanocrystals or read the Molecular Probes® Handbook Section 6.6—Qdot® Nanocrystals to find out more.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Alexa Fluor™ 568 Protein Labeling Kit (Invitrogen™)

Molecular Probes® Protein Labeling Kits provide a convenient means for attaching a fluorescent label (or biotin) to an antibody (or a protein larger than 40 kDa). Conjugates are ideal for multiple applications, including flow cytometry, fluorescent microscopy, immunohistochemistry, primary detection, ELISAs, immunocytochemistry, FISH, and more. Kits are available in 12 Alexa Fluor® dye colors, biotin, the hapten Oregon Green® 488, fluorescein EX, and Texas Red® dye. Each kit provides the components needed to perform three protein conjugations and purifications.

Important Features of Protein Labeling Kits:

• Labeled proteins typically ready to use in 2 hr (~30 min hands-on time)
• Designed to label 1 mg of IgG
• Simple protocol — react, separate, use
• Stabilizing proteins must be removed from the sample before labeling


The Benefits of Alexa Fluor® Dyes
Compared to traditional dyes, Alexa Fluor® dyes are brighter, more photostable, and more pH resistant between pH 4 and 10. And generally when using Alexa Fluor® dyes, higher degrees of labeling can be achieved without intramolecular quenching. For details see Alexa Fluor® Dyes Spanning the Visible and Infrared Spectrum—Section 1.3.

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices. To learn more about our various kits read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in the Molecular Probes® Handbook.

We’ll Make a Custom Antibody Conjugate for You
If you can’t find what you’re looking for in our stocked list, we’ll prepare a custom antibody conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

Alexa Fluor™ 647 Cadaverine (Invitrogen™)

Alexa Fluor® 647 Cadaverine is useful as a polar tracer and as a reactive dye for labeling proteins via a carboxylic acid moiety. Alexa Fluor® 647 is a bright, far red dye with excitation ideally suited to the 633 nm laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 647 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 647 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

Detailed information about this AlexaFluor® cadaverine:

• Fluorophore label : Alexa Fluor® 647 dye
• Reactive group: cadaverine
• Reactivity: carboxylic acids, aldehydes, and ketones (and glutamine residues through an enzyme-catalyzed transamidation reaction)
• Ex/Em of the conjugate: 651/672 nm
• Extinction coefficient: 245,000 cm-1M-1
• Spectrally similar dyes: APC, Cy5
• Molecular weight: ~1000

Cell Tracking and Tracing Applications
Alexa Fluor® cadaverines make excellent fluorescent polar tracers because they are bright, small, and water soluble. Since they contain an aldehyde-fixable functional group, they can be fixed in cells by treatment with formaldehyde or glutaraldehyde. They are easily loaded into cells by microinjection, infusion from patch pipette, or uptake induced by our Influx™ Pinocytic Cell-Loading Reagent. Learn more about cell tracking and tracing.

Protein Labeling Applications
Alexa Fluor® cadaverines can be used as reactive molecules for adding a fluorescent label to carboxylic acids using a coupling agent such as a carbodiimide; they do not spontaneously react with carboxylic acids in solution. They do, however, react spontaneously with the common amine-reactive functional groups, including succinimidyl esters and isothiocyanates. The amine-containing Alexa Fluor® cadaverines can also be used to label glutamine residues in some proteins and peptides via an enzyme-catalyzed transamidation reaction.

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

Related Products
DMSO (dimethylsulfoxide) (D12345)
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

DyLight™ 755 Antibody Labeling Kit (Thermo Scientific™)

The Thermo Scientific DyLight 755 Antibody Labeling Kit contains an NHS ester-activated derivative of high-performance DyLight 755 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods. The standard size kit contains all necessary components to perform three separate labeling reactions using 1 mg of IgG or similar quantities of other proteins—the amine-reactive DyLight 755 NHS-ester in convenient single-use vials as well as purification resin and spin columns for the preparation of ready-to-use conjugate.

DyLight 755 is a near-IR fluor that is invisible to the naked eye but increases the staining options when using infrared imaging systems. DyLight 755 has spectral properties that are very similar to other near-IR dyes, including Alexa Fluor™ 750. The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates. DyLight 755 Amine-Reactive Dye is also available as a stand-alone reagent.

Features of DyLight 755 NHS Ester:

High performance—DyLight 755 replaces Alexa Fluor 755 for near-infrared staining
Specific—NHS ester-activated dye labels proteins and other molecules at primary amines (-NH2)
Convenient kit sizes—standard and microscale sizes are offered to match your experimental needs
Optimized procedure—following the standard protocol results in antibodies with excellent dye:protein ratios and recovery rates for optimum activity and fluorescence labeling

Applications:
• Primary antibody labeling for immunofluorescence microscopy, immunohistochemistry (IHC), Western blotting, or ELISA assay
• Target protein labeling for in vitro and in vivo fluorescent detection strategies

DyLight 755 Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

Typical labeling reactions require the dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to specific amounts of the labeling reagent. This feature allows DyLight 755 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

Related Products
DyLight™ 755 NHS Ester
DyLight™ 755 Microscale Antibody Labeling Kit

Zenon™ R-Phycoerythrin Human IgG Labeling Kit (Invitrogen™)

Zenon labeling technology provides a fast, versatile and reliable method for producing antibody conjugates, even with very small (submicrogram) amounts of starting material. Antibody conjugates formed using Zenon technology may be used to stain cells in any protocol where a directly labeled primary antibody is suitable, including flow cytometry, imaging, high throughput and other applications. Moreover, this technology simplifies applications that previously were time consuming or not practical, such as the use of multiple mouse-derived antibodies in the same staining protocol.

View a selection guide for all Zenon™ antibody labeling kits and other antibody labeling products.

Click-iT™ Plus OPP Alexa Fluor™ 488 Protein Synthesis Assay Kit (Invitrogen™)

The Click-iT® Plus OPP Alexa Fluor® 488 Protein Synthesis Assay Kit provides a fast, sensitive, and non-radioactive method for the detection of protein synthesis using fluorescence microscopy or high-content imaging. In this assay O-propargyl-puromycin (OPP) is efficiently incorporated into newly translated proteins in complete methionine-containing media and fluorescently labeled with a bright, photostable Alexa Fluor® dye in a fast, highly-specific, and mild click reaction.

Features of the kit include:

• No media change required—works in complete, methionine-containing media, no need to remove cell media
• Multiplex-enabled—Click-iT® Plus technology retains signal from GFP and binding of fluorescent-conjugated phalloidins
• Non-radioactive—an alternative to the traditional 35S-methionine methods
• Works in vivo—published results demonstrate use in vivo for determination of protein translation

The kit contains O-propargyl-puromycin (OPP), which is an alkyne analog of puromycin (also available separately), as well as Alexa Fluor® 488 picolyl azide and all necessary reagents to perform the Click reaction. The OPP is fed to cultured cells and incorporated into proteins during active protein synthesis. Addition of the Alexa Fluor® 488 picolyl azide and the Click reaction reagents leads to a chemoselective ligation, or "click" reaction, between the picolyl azide dye and the OPP alkyne, allowing the modified proteins to be detected by imaged-based analysis.

The click reaction uses bioorthogonal (biologically unique) moieties to fluorescently label proliferating cells, helping produce low backgrounds and high detection sensitivities. Because of the mild reaction conditions, Click-iT® Plus assays detect protein translation events while enabling the preservation of cell morphology, the binding of fluorescently-labeled phalloidin, and the fluorescent signal from GFP.

Unlike 35S-methionine, used in traditional methods, OPP is not an amino acid analog, so it can be added directly to cells in complete media or used to determine protein synthesis in vivo.

The kit contains all of the components needed to label and detect the incorporated OPP in newly translated proteins in samples of adherent cells. The kit includes sufficient reagents for the labeling of 25 18 mm × 18 mm coverslips using 1 mL of reaction buffer per test.

Alexa Fluor™ 750 NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor® 750 is a bright and photostable near-IR dye that is spectrally similar to Cy7. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 750 dye is water soluble and pH-insensitive from pH 4 to pH 10. Fluorescence of this long-wavelength Alexa Fluor® dye is not visible to the human eye but is readily detected by most imaging systems. In addition to reactive dye formulations, we offer Alexa Fluor® 750 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

The NHS ester (or succinimidyl ester) of Alexa Fluor® 750 is the most popular tool for conjugating this dye to a protein or antibody. NHS esters can be used to label to the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting Alexa Fluor® conjugate will exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores.

Detailed information about this AlexaFluor® NHS ester:

Fluorophore label: Alexa Fluor® 750 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 753/782 nm
Extinction coefficient: 290,000 cm-1M-1
Spectrally similar dyes: Cy7
Molecular weight: ~1300

Typical Conjugation Reaction
You can conjugate amine-reactive reagents with virtually any protein or peptide (the provided protocol is optimized for IgG antibodies). You can scale the reaction for any amount of protein, but the concentration of the protein should be at least 2 mg/mL for optimal results. We recommend trying three different degrees of labeling, using three different molar ratios of the reactive reagent to protein.

The Alexa Fluor® NHS ester is typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO) (D12345), and the reaction is carried out in 0.1–0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature for 1 hour. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, you may achieve more selective labeling of the amine terminus using a buffer closer to neutral pH.

Conjugate Purification
Labeled antibodies are typically separated from free Alexa Fluor® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

Dabcyl, SE, 4-((4-(dimethylamino)phenyl)azo)benzoic Acid, Succinimidyl Ester (Invitrogen™)

The amine-reactive quencher, dabcyl succinimidyl ester has a broad and intense visible absorption but no fluorescence making it useful as an acceptor in fluorescence resonance energy transfer (FRET) applications.

Alexa Fluor™ 647 C2 Maleimide (Invitrogen™)

Alexa Fluor® 647 is a bright, far-red fluorescent dye with excitation ideally suited for the 594 nm or 633 nm laser lines. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 647 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 647 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

The maleimide derivative of Alexa Fluor® 647 is the most popular tool for conjugating the dye to a thiol group on a protein, oligonucleotide thiophosphate, or low molecular weight ligand. The resulting Alexa Fluor® 647 conjugates exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores.

Detailed information about this AlexaFluor® maleimide:

Fluorophore label: Alexa Fluor® 647 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 651/671 nm
Extinction coefficient: 265,000 cm-1M-1
Spectrally similar dyes: Cy5
Molecular weight: ~1250

Typical Conjugation Reaction
The protein should be dissolved at a concentration of 50-100 µM in a suitable buffer (10-100 mM phosphate, Tris, or HEPES) at pH 7.0-7.5. In this pH range, the protein thiol groups are sufficiently nucleophilic that they react almost exclusively with the reagent in the presence of the more numerous protein amine groups, which are protonated and relatively unreactive. We recommend reducing any disulfide bonds at this point using a 10-fold molar excess of reducing agent such as DTT or TCEP. Excess DTT must be removed by dialysis and subsequent thiol-modification should be carried out under oxygen-free conditions to prevent reformation of the disulfide bonds; these precautions are not necessary when using TCEP prior to maleimide conjugation.

The Alexa Fluor® maleimide is typically dissolved in high-quality anhydrous dimethylsulfoxide (DMSO) at a concentration of 1-10 mM immediately prior to use, and stock solutions should be protected from light as much as possible. Generally, this stock solution is added to the protein solution dropwise while stirring to produce approximately 10-20 moles of reagent per mole of protein, and the reaction is allowed to proceed at room temperature for 2 hours or at 4°C overnight, protected from light. Any unreacted thiol-reactive reagent can be consumed by adding excess glutathione, mercaptoethanol, or other soluble low molecular weight thiol.

Conjugate Purification
Labeled antibodies are typically separated from free Alexa Fluor® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

Qtracker™ 605 Cell Labeling Kit (Invitrogen™)

The reagents in the Qtracker® 605 Cell Labeling Kit use a custom targeting peptide to deliver orange-fluorescent Qdot® 605 nanocrystals into the cytoplasm of live cells. Qtracker® Cell Labeling Kits are designed for loading cells grown in culture with highly fluorescent Qdot® nanocrystals. Once inside the cells, Qtracker® labels provide intense, stable fluorescence that can be traced through several generations, and are not transferred to adjacent cells in a population.

Need a different emission spectrum or longer tracking? View our other mammalian cell tracking products.

Key Attributes:

Qtracker® 605 label has Ex/Em (405-565/605) nm
Designed for loading cells grown in culture with highly fluorescent Qdot® nanocrystals
Provide intense, stable fluorescence that can be traced through several generations
Available in eight colors—525 nm, 565 nm, 585 nm, 605 nm, 625 nm, 655 nm, 705 nm, or 800 nm emission
Excellent tools for long-term tracking or imaging studies of live cells, including migration, motility, morphology, and other cell function assays

The Qtracker® Cell Labeling Kits use a custom targeting peptide to deliver Qdot® nanocrystals into the cytoplasm of live cells. Cytoplasmic delivery by this mechanism is not mediated by a specific enzyme; therefore, no cell-type specificity has been observed. Delivery is typically accomplished in less than 1 hour.

Qdot® nanocrystals delivered by the Qtracker® Cell Labeling Kits are compatible with serum-sensitive cells; intense fluorescence is maintained in complex cellular environments and under various biological conditions including changes in intracellular pH, temperature, and metabolic activity. Furthermore, autofluorescence commonly observed in cells or tissues can be avoided using Qtracker® 655, 705, or 800 Kits.

Long-Lasting, Targeted Signal
Using Qtracker® Cell Labeling Kits, you can observe labeled cells using extensive continuous illumination, without the photobleaching and degradation problems often associated with organic dyes. Qtracker® labels are distributed in vesicles in the cytoplasm, and are inherited by daughter cells for at least six generations. Fluorescence from the Qtracker® labels can be seen up to a week after delivery in some cell lines. Long-term cellular retention makes Qtracker® Cell Labeling Kits ideal for studying cell motility, migration, differentiation, morphology, and many other cellular function studies. Qtracker® labels do not leak out of intact cells to be taken up by adjacent cells in the population.

Monitor the Signal on Multiple Platforms
Qtracker® reagent-labeled live cells can be easily monitored on a variety of platforms, including flow cytometry, fluorescence/confocal microscopy, fluorescence microplate readers, and high-content imaging systems.

Minimal Impact on Live Cells
The cytotoxicity of the materials use in Qtracker® Cell Labeling Kits has been tested in a variety of cell lines including CHO, HeLa, U-118, 3T3, HUVEC, and Jurkat cells. Labeling with Qtracker® Cell Labeling Kits appears to exert minimal impact on cellular surface marker expression, cell proliferation, cellular enzyme activity, and cell motility.

Useful in a Variety of Cell Tracing Studies
Post-labeling, researchers have demonstrated a wide variety of applications for Qtracker® labeled cells, including cell co-culture and cell assembly into heterotypic assemblies, multilineage differentiation, trans-differentiation versus cell fusion, embryonic and mesenchymal stem cell tracking, and cell migration dynamics.

DyLight™ 488 Antibody Labeling Kit (Thermo Scientific™)

The Thermo Scientific DyLight 488 Antibody Labeling Kit contains an NHS ester-activated derivative of high-performance DyLight 488 for fluorescent labeling of antibodies and other proteins to be used as molecular probes for cellular imaging and other fluorescence detection methods. The standard size kit contains all necessary components to perform three separate labeling reactions using 1 mg of IgG or similar quantities of other proteins.

DyLight 488 has high fluorescence intensity over a broad pH range (pH 4-9) and is more photostable than Cy2™, Alexa™ Fluor 488, FITC and LI-COR™ dyes in many applications. The high water solubility of DyLight Fluors allows a high dye-to-protein ratio to be achieved without causing precipitation of the conjugates. DyLight 488 Amine-Reactive Dye is also available as a stand-alone reagent.

Features of the DyLight 488 NHS Ester:

High performance— DyLight 488 is comparable to Alexa Fluor 488 and brighter than FITC and Cy2
Specific— NHS ester-activated dye labels proteins and other molecules at primary amines (-NH2)
Convenient kit sizes— standard and microscale sizes are offered to match your experimental needs
Optimized procedure— following the standard protocol results in antibodies with excellent dye:protein ratios and recovery rates for optimum activity and fluorescence labeling

Applications:
• Primary antibody labeling for immunofluorescence microscopy, immunohistochemistry (IHC), Western blotting or ELISA assay
• Target protein labeling for in vitro and in vivo fluorescent detection strategies

DyLight 488 Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

Typical labeling reactions require the dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to specific amounts of the labeling reagent. This feature allows DyLight 488 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

We also offer Standard and Microscale DyLight 488 Antibody Labeling Kits for fast and efficient fluorescent labeling of antibodies for use in fluorescence methods. The standard size kit contains all necessary components to perform three separate labeling reactions using 1 mg of IgG or similar quantities of other proteins. The microscale kit contains all of the necessary components to perform five separate labeling reactions using 100 µg of IgG. Both kit sizes include the Amine-Reactive DyLight 488 NHS-ester in convenient single-use vials as well as purification resin and spin columns for the preparation of ready-to-use conjugate.

Related Products
DyLight™ 488 NHS Ester
DyLight™ 488 Microscale Antibody Labeling Kit

Oregon Green™ 488 Carboxylic Acid, Succinimidyl Ester, 5-isomer (Invitrogen™)

This single 5-isomer preparation of our proprietary, amine-reactive dye, Oregon Green 488 carboxylic acid, succinimidyl ester and its conjugates have green fluorescence similar to that of fluorescein but are more photostable. Additionally, Oregon Green 488 dye has a lower pKa than fluorescein (pKa = 4.7 versus 6.4 for fluorescein), making this bright dye less pH sensitive in the physiological range.

Texas Red™ C2-Dichlorotriazine (Invitrogen™)

Texas Red C2-dichlorotriazine is a reactive dye with absorption/emission maxima of ~588/601 nm. Dichlorotriazines readily modify amines in proteins, and are among the few reactive groups that are reported to react directly with polysaccharides and other alcohols in aqueous solution, provided that the pH is >9 and that other nucleophiles are absent.

FITC (5/6-fluorescein isothiocyanate), mixed isomer (Thermo Scientific™)

Fluorescein isothiocyanate (FITC) is an amine-reactive derivative of fluorescein dye that has wide-ranging applications as a label for antibodies and other probes, for use in fluorescence microscopy, flow cytometry and immunofluorescence-based assays such as Western blotting and ELISA. The isothiocyanate variety of fluorescein efficiently labels antibodies and other purified proteins at primary amines (lysine side chains)

Properties of FITC:

• Alternative names: 5/6-FITC
• Chemical name: 5(6)-fluorescein isothiocyanate mixed isomer
• Molecular weight: 389.2
• Excitation source: 488 nm spectral line, argon-ion laser
• Excitation wavelength: 494 nm
• Emission wavelength: 518 nm
• Extinction coefficient: > 70,000 M-1cm-1
• CAS #: 27072-45-3
• Purity: > 95% by HPLC
• Solubility: Soluble in aqueous buffers at pH > 6
• Reactive groups: Isothiocyanate, reacts with primary amines at pH 7.0 to 9.0

Applications
• Label antibodies for use as immunofluorescent probes
• Label oligonucleotides for hybridization probes
• Detect proteins in gels and on Western blots

Related Products
NHS-Fluorescein (5/6-carboxyfluorescein succinimidyl ester), mixed isomer
Pierce™ NHS-Fluorescein Antibody Labeling Kit
Pierce™ FITC Antibody Labeling Kit

Alexa Fluor™ 568 C5 Maleimide (Invitrogen™)

Alexa Fluor® 568 is a bright, orange/red fluorescent dye with excitation ideally suited for the 568 nm laser line on the Ar-Kr mixed-gas laser. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 568 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 568 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

The maleimide derivative of Alexa Fluor® 568 is the most popular tool for conjugating the dye to a thiol group on a protein, oligonucleotide thiophosphate, or low molecular weight ligand. The resulting Alexa Fluor® 568 conjugates exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores.

Detailed information about this AlexaFluor® maleimide:

Fluorophore label: Alexa Fluor® 568 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 575/600 nm
Extinction coefficient: 92,000 cm-1M-1
Spectrally similar dyes: Rhodamine red
Molecular weight: 880.92

Typical Conjugation Reaction
The protein should be dissolved at a concentration of 50-100 µM in a suitable buffer (10-100 mM phosphate, Tris, or HEPES) at pH 7.0-7.5. In this pH range, the protein thiol groups are sufficiently nucleophilic that they react almost exclusively with the reagent in the presence of the more numerous protein amine groups, which are protonated and relatively unreactive. We recommend reducing any disulfide bonds at this point using a 10-fold molar excess of reducing agent such as DTT or TCEP. Excess DTT must be removed by dialysis and subsequent thiol-modification should be carried out under oxygen-free conditions to prevent reformation of the disulfide bonds; these precautions are not necessary when using TCEP prior to maleimide conjugation.

The Alexa Fluor® maleimide is typically dissolved in high-quality anhydrous dimethylsulfoxide (DMSO) at a concentration of 1-10 mM immediately prior to use, and stock solutions should be protected from light as much as possible. Generally, this stock solution is added to the protein solution dropwise while stirring to produce approximately 10-20 moles of reagent per mole of protein, and the reaction is allowed to proceed at room temperature for 2 hours or at 4°C overnight, protected from light. Any unreacted thiol-reactive reagent can be consumed by adding excess glutathione, mercaptoethanol, or other soluble low molecular weight thiol.

Conjugate Purification
Labeled antibodies are typically separated from free Alexa Fluor® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

Alexa Fluor™ 532 C5 Maleimide (Invitrogen™)

Alexa Fluor® 532 is a bright, yellow fluorescent dye with excitation ideally suited for the frequency-doubled Nd:YAG laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 532 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 532 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

The maleimide derivative of Alexa Fluor® 532 is the most popular tool for conjugating the dye to a thiol group on a protein, oligonucleotide thiophosphate, or low molecular weight ligand. The resulting Alexa Fluor® 532 conjugates exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores.

Detailed information about this AlexaFluor® maleimide:

Fluorophore label: Alexa Fluor® 532 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 528/552 nm
Extinction coefficient: 78,000 cm-1M-1
Molecular weight: 812.88

Typical Conjugation Reaction
The protein should be dissolved at a concentration of 50-100 µM in a suitable buffer (10-100 mM phosphate, Tris, or HEPES) at pH 7.0-7.5. In this pH range, the protein thiol groups are sufficiently nucleophilic that they react almost exclusively with the reagent in the presence of the more numerous protein amine groups, which are protonated and relatively unreactive. We recommend reducing any disulfide bonds at this point using a 10-fold molar excess of reducing agent such as DTT or TCEP. Excess DTT must be removed by dialysis and subsequent thiol-modification should be carried out under oxygen-free conditions to prevent reformation of the disulfide bonds; these precautions are not necessary when using TCEP prior to maleimide conjugation.

The Alexa Fluor® maleimide is typically dissolved in high-quality anhydrous dimethylsulfoxide (DMSO) at a concentration of 1-10 mM immediately prior to use, and stock solutions should be protected from light as much as possible. Generally, this stock solution is added to the protein solution dropwise while stirring to produce approximately 10-20 moles of reagent per mole of protein, and the reaction is allowed to proceed at room temperature for 2 hours or at 4°C overnight, protected from light. Any unreacted thiol-reactive reagent can be consumed by adding excess glutathione, mercaptoethanol, or other soluble low molecular weight thiol.

Conjugate Purification
Labeled antibodies are typically separated from free Alexa Fluor® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

Alexa Fluor™ 488 TFP ester (Invitrogen™)

Alexa Fluor® dyes are reactive molecules that can be used to add a fluorescent label to the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting Alexa Fluor® conjugates exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores. Alexa Fluor® 488 TFP ester produces a conjugate with excitation/emission of 495/515 nm that is spectrally similar to fluorescein (FITC) and Cy2 conjugates.

Alexa Fluor® dyes are available with fluorescence emissions that span the visible and near-infrared spectrum (see The Alexa Fluor® Dye Series—Note 1.1 in The Molecular Probes® Handbook) and provide the unique combination of water solubility and pH insensitivity between pH 4 and 10 for compatibility in diverse biological environments.

TFP (tetrafluorophenyl) esters are an improvement over the succinimidyl ester (SE or NHS-ester) chemistry typically used to attach fluorophores or haptens to the primary amines of biomolecules. Both reactive chemistries produce the same strong amide bond between the dye or hapten and the compound of interest, but TFP esters are less susceptible to spontaneous hydrolysis during conjugation reactions. Alexa Fluor® TFP esters are stable for several hours at the basic pH typically used for reactions–far outlasting succinimidyl esters.

Typical Conjugation Reaction
You can conjugate amine-reactive reagents with virtually any protein or peptide; the provided protocol is optimized for IgG antibodies. You may scale the reaction for any amount of protein, but the concentration of the protein should be at least 2 mg/mL for optimal results. We recommend trying three different degrees of labeling, using three different molar ratios of the reactive reagent to protein.

The Alexa Fluor® TFP ester is typically dissolved in high-quality, anhydrous dimethylsulfoxide (DMSO) (D12345) , and the reaction is carried out in 0.1–0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature for 1 hour. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, you may achieve more selective labeling of the amine terminus using a buffer closer to neutral pH.

Conjugate Purification
Labeled antibodies are typically separated from free Alexa Fluor® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis.

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we are ISO 9001:2000 certified.

Oregon Green™ 488 Azide (Oregon Green™ 6-Carboxamido-(6-Azidohexanyl), Triethylammonium Salt), 6-isomer (Invitrogen™)

The green-fluorescent Oregon Greenr® 488 azide is reactive with terminal alkynes via a copper-catalyzed click reaction. In addition to being a bright and photostable alternative to fluorescein for use in flow cytometry, microscopy and HCS, Oregon Green® 488 can also be utilized as a bio-orthogonal or biologically unique hapten for use in applications requiring signal amplification.

DyLight™ 350 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 350 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 350 used to fluorescently label sulfhydryl-containing peptides, proteins and other biomolecular probes.

DyLight 350 has high fluorescence intensity over a broad pH range (pH 4-9) and is more photostable than Alexa Fluor™ 350 and AMCA dyes in many applications. The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates.

Features of DyLight 350 Sulfhydryl-Reactive Dye:

High performance—DyLight 350 shows brighter fluorescence than Alexa Fluor 350 and AMCA
Specific—maleimide-activated dye labels proteins and other molecules at reduced sulfhydryls (-SH)
Efficient labeling methods—well-characterized chemistry and optimized protocols provide for reliable, high-quality labeling
Optimized antibody labeling procedure—complete protocol for IgG reduction and labeling and calculating the labeling efficiency

Applications:
• Antibody labeling for immunofluorescence applications, including immunocytochemistry (ICC), immunohistochemistry (IHC), Western blotting and ELISA assay
• Target macromolecule labeling for in vitro and in vivo fluorescent detection strategies

DyLight 350 Sulfhydryl-Reactive Dye is activated with a maleic acid imide (maleimide) moiety to form a reactive alkylation reagent. Labeling occurs through reaction of the maleimide-activated dye with reduced sulfhydryl groups (-SH) to form stable thioether bonds. Maleimides are specific for sulfhydryl groups between pH 6.5-7.5. Learn more about maleimide chemistry.

Related Products
DyLight™ 350 Maleimide

Alexa Fluor™ 488 Cadaverine (Invitrogen™)

Alexa Fluor® 488 Cadaverine is useful as a polar tracer and as a reactive dye for labeling proteins via a carboxylic acid moiety. Alexa Fluor® 488 is a bright, green-fluorescent dye with excitation ideally suited to the 488 nm laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 488 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 488 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

Detailed information about this AlexaFluor® cadaverine:

• Fluorophore label : Alexa Fluor® 488 dye
• Reactive group: cadaverine
• Reactivity: carboxylic acids, aldehydes, and ketones (and glutamine residues through an enzyme-catalyzed transamidation reaction)
• Ex/Em of the conjugate: 493/516 nm
• Extinction coefficient: 73,000 cm-1M-1
• Spectrally similar dyes: FITC, GFP
• Molecular weight: 640.61

Cell Tracking and Tracing Applications
Alexa Fluor® cadaverines make excellent fluorescent polar tracers because they are bright, small, and water soluble. Since they contain an aldehyde-fixable functional group, they can be fixed in cells by treatment with formaldehyde or glutaraldehyde. They are easily loaded into cells by microinjection, infusion from patch pipette, or uptake induced by our Influx™ Pinocytic Cell-Loading Reagent. Learn more about cell tracking and tracing.

Protein Labeling Applications
Alexa Fluor® cadaverines can be used as reactive molecules for adding a fluorescent label to carboxylic acids using a coupling agent such as a carbodiimide; they do not spontaneously react with carboxylic acids in solution. They do, however, react spontaneously with the common amine-reactive functional groups, including succinimidyl esters and isothiocyanates. The amine-containing Alexa Fluor® cadaverines can also be used to label glutamine residues in some proteins and peptides via an enzyme-catalyzed transamidation reaction.

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

Related Products
DMSO (dimethylsulfoxide) (D12345)
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

BODIPY™ 576/589 NHS Ester (Succinimidyl Ester) (Invitrogen™)

BODIPY® 576/589 dye is bright, red fluorescent dye with similar excitation and emission to Rhodamine Red™ and Alexa Fluor®568. It has a high extinction coefficient and fluorescence quantum yield and is relatively insensitive to solvent polarity and pH change. In contrast to the highly water soluble fluorophores Alexa Fluor® 488 dye and fluorescein (FITC), BODIPY® dyes have unique hydrophobic properties ideal for staining lipids, membranes, and other lipophilic compounds. BODIPY® 576/589 dye has a relatively long excited-state lifetime (typically 5 nanoseconds or longer), which is useful for fluorescence polarization-based assays and a large two-photon cross-section for multiphoton excitation. In addition to reactive dye formulations, we offer BODIPY® 576/589 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection.

The NHS ester (or succinimidyl ester) of BODIPY® 576/589 is the most popular tool for conjugating the dye to a protein or antibody. NHS esters can be used to label the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting BODIPY® 576/589 conjugates exhibit bright fluorescence, narrow emission bandwidths, and relatively long excited-state lifetimes, which can be useful for fluorescence polarization assays and two-photon excitation (TPE) microscopy.

This reactive dye contains a C3 alkyl spacer between the fluorophore and the NHS ester group. This spacer helps to separate the fluorophore from its point of attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated.

Detailed information about this BODIPY® 576/589 NHS ester:

Fluorophore label: BODIPY® 576/589 dye
Reactive group: NHS ester (succinimidyl ester)
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 575/588 nm
Extinction coefficient: 83,000 cm-1M-1
Molecular weight: 426.19

Typical Conjugation Reaction
Amine-reactive reagents can be conjugated with virtually any protein or peptide; the provided protocol is optimized for IgG antibodies. The reaction can be scaled for any amount of protein, but the concentration of the protein should be at least 2 mg/mL for optimal results. We recommend trying three different degrees of labeling, using three different molar ratios of the reactive reagent to protein.

The BODIPY® NHS ester is typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO), and the reaction is carried out in 0.1-0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature for 1 hour. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, you may achieve more selective labeling of the amine terminus using a buffer closer to neutral pH.

Conjugate Purification
Labeled antibodies are typically separated from free BODIPY® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration medium with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

Zenon™ Alexa Fluor™ 647 Rabbit IgG Labeling Kit (Invitrogen™)

Zenon® labeling technology provides a fast, versatile, and reliable method for adding a fluorescent label to an antibody. You need only a small amount of starting material, and the method is optimized for efficient labeling of antibodies in serum, ascites fluid, or hybridoma suspensions. Antibody conjugates formed using Zenon® technology may be used in any protocol where a directly labeled primary antibody is suitable, including flow cytometry, imaging, and high-throughput applications. This exclusive Molecular Probes® Zenon® labeling technology greatly simplifies the use of multiple mouse-derived antibodies in the same staining protocol.

Important Features of Zenon® Labeling Technology:

• Labeled antibodies typically ready to use in 10 minutes
• Requires only 1–20 μg primary antibody
• Simple, no purification required
• Flexible–over 24 fluorophores plus biotin, HRP, alkaline phosphatase, and TSA to choose from
• Multiplex with other mouse monoclonal antibodies simultaneously


Save Time and Antibody
Each kit comes with affinity-purified monovalent Fab fragment of a goat anti-Fc antibody (or, in the case of the Zenon® Goat IgG Labeling Kits, a rabbit anti-Fc antibody) that has been conjugated to one of our premier Alexa Fluor® dyes or to Pacific Blue™, Pacific Orange™, fluorescein, or Texas Red®-X dyes, biotin R-phycoerythrin (R-PE), allophycocyanin (APC), HRP, or alkaline phosphatase.

Formation of the Fab–antibody complex with the Zenon® Antibody Labeling Kits is extremely fast (5 min for complex, 5 min for blocking step). And Zenon® labeling is a reliable and reproducible method, even with as low 0.4 μg in 2 μL of primary antibody. There is minimal waste of expensive or difficult-to-obtain antibodies when using the Zenon® Antibody Labeling Kits.

Preserve Primary Antibody Function and Affinities
Reactive dye labeling of primary antibodies can have unpredictable and undesirable outcomes. Among these are reduced binding affinities by label addition in the binding pocket. Zenon® antibody labeling approach, targeted to the Fc tail, avoids this concern.

Moreover the Zenon® dye- and enzyme-labeled Fab fragments have been affinity purified during their preparation to help ensure their high affinity and selectivity for the Fc portion of the corresponding primary antibody. The procedure for chemical labeling of the Fab fragments protects the Fc-binding site, resulting in more active labeling reagents.

Many Fluorophore and Enzyme Labels Available
Zenon® immunolabeling technology makes it very easy to change fluorescent color combinations or detection methodologies by simply using a different dye- or enzyme-labeled Fab fragment from our extensive selection of over 100 Zenon® Antibody Labeling Kits. If larger quantities or covalent attachment of the label is desired, see Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices.

Zenon® Technology Simplifies the Use of Multiple Antibodies of the Same Isotype in the Same Protocol
The stability of the Zenon® complex is sufficient to allow sequential (or simultaneous) labeling of different targets in cells and tissues with multiple antibody complexes. Subsequent to staining, an aldehyde-based fixation step can permanently block the transfer of Zenon® labels between different primary antibodies and will preserve the staining pattern.

We’ll Make a Custom Antibody Conjugate for You
If you can’t find what you’re looking for in our stocked list, we’ll prepare a custom antibody conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

Related Links:

Zenon® Labeling Technology
Zenon® Technology: Versatile Reagents for Immunolabeling—Section 7.3

Zenon™ Allophycocyanin Rabbit IgG Labeling Kit (Invitrogen™)

Zenon labeling technology provides a fast, versatile and reliable method for producing antibody conjugates, even with very small (submicrogram) amounts of starting material. Antibody conjugates formed using Zenon technology may be used to stain cells in any protocol where a directly labeled primary antibody is suitable, including flow cytometry, imaging, high throughput and other applications. Moreover, this technology simplifies applications that previously were time consuming or not practical, such as the use of multiple mouse-derived antibodies in the same staining protocol.

View a selection guide for all Zenon™ antibody labeling kits and other antibody labeling products.

Qdot™ 545 ITK™ Carboxyl Quantum Dots (Invitrogen™)

Qdot® 545 ITK™ carboxyl quantum dots are the ideal starting material for preparing custom conjugates that require high loading of biomolecules. These materials are carboxylate functionalized and can be coupled to amine groups of proteins and modified oligonucleotides using EDC-mediated condensation. The coatings of these probes provides more binding sites than our Qdot® ITK™ amino quantum dots, but lacks PEG linkers that help to prevent non-specific interactions. These materials can be conjugated to X-PEG-amine bi-functional linkers for custom reactivity and higher specificity. Our Qdot® ITK™ carboxyl quantum dots are provided as 8 µM solutions and are available in all 9 Qdot® probe colors.

Important Features of Qdot® ITK™ Carboxyl Quantum Dots:
• Qdot® 545 ITK™ carboxyl quantum dot has emission maxima of ~545 nm
• Extremely photostable and bright fluorescence
• Efficiently excited with single-line excitation sources
• Narrow emission, large Stokes shift
• Available in multiple colors
• Ideal labeling and tracking applications


Properties of Qdot® Nanocrystals
Qdot® probes are ideal for imaging and labeling applications that require bright fluorescent signals and/or real-time tracking. Unique among fluorescent reagents, all nine available colors of Qdot® probes can be simultaneously excited with a single (UV to blue-green) light source. This property makes these reagents excellent for economical and user-friendly multiplexing applications. Qdot® labels are based on semiconductor nanotechnology and are similar in scale to moderately sized proteins.

About the Innovator’s Tool Kit Qdot® ITK™ Reagents
These Qdot® ITK™ probes are ideal for researchers who wish to prepare specific (non-stocked) conjugates for their applications and need customizable conjugation functionality.

Other Forms of Qdot® Nanocrystals are Available
In addition to the carboxyl-derivatized form, we offer Qdot® ITK™ quantum dots with amino and aliphatic hydrocarbon modifications. We’ve also developed a wide range of Qdot® nanocrystals conjugates and labeling kits. Investigate the properties of Qdot® nanocrystals or read the Molecular Probes® Handbook Section 6.6—Qdot® Nanocrystals to find out more.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Biotin-X, SE (6-((Biotinoyl)Amino)Hexanoic Acid, Succinimidyl Ester (Biotinamidocaproate, N-Hydroxysuccinimidyl Ester)) (Invitrogen™)

The amine-reactive biotin-X, SE can be used to attach this important hapten to biomolecules of interest for subsequent detection with streptavidin, avidin or NeutrAvidin® biotin-binding protein.

EZ-Link™ Plus Activated Peroxidase (Thermo Scientific™)

Thermo Scientific Pierce Plus Activated Peroxidase is an amine-reactive form of horseradish peroxidase (HRP) that provides coupling efficiencies of greater than 95% with antibodies and other proteins. This product consists of 5 mg of lyophilized HRP and is sufficient to modify 5 mg of Immunoglobulin (IgG)

Features of Thermo Scientific Pierce Plus Activated Peroxidase:

Activated HRP – periodate-treated, aldehyde-activated horseradish peroxidase, ready for conjugation to antibodies and other proteins at sites of primary amines (e.g., lysines)
Permanent conjugation – reacts efficiently (95%) with primary amines to form covalent amide bonds upon treatment with sodium cyanoborohydride (included in kit)
High activity HRP – enzyme activity is 120 to 200 units/mg; lyophilized, activated enzyme is stable for at least 12 months at -20°C
Convenient quantities – each 1 mg-quantity of activated enzyme is sufficient for reaction with 1 mg of IgG to produce about 0.5 mL of conjugate
Customizable – vary the molar ratios, reaction buffer and pH, and other parameters to acheive conjugates with different levels of HRP incorporation and activity

Plus Activated Peroxidase is horseradish peroxidase (HRP) enzyme, whose native carbohydrates (sugars) have been gently oxidized with periodate to produce amine-reactive aldehyde groups. These carbonyls of Plus Activated Peroxidase spontaneously and efficiently crosslink with primary amines on antibody or other proteins. The method is more effective than other amine-reactive chemistries, such as glutaraldehyde coupling, which often causes polymerization and a greater degree of conjugate inactivation. The pre-activated enzyme eliminates the difficulties inherent in preparing and validating activated peroxidase from scratch. The kit provides the enzyme, accessory reagents and protocol to easily produce high-performance conjugates for Western blotting, ELISA and other detection techniques.

Related Products
EZ-Link™ Plus Activated Peroxidase Kit

Qdot™ 655 Biotin Conjugate Kit (Invitrogen™)

The biotin-labeled Qdot® 655 nanocrystals are available for detecting streptavidin probes or for creating noncovalent conjugates with streptavidin-labeled molecules or with other biotinylated molecules using a streptavidin bridge. The product is provided as 250 µL of a 2 µM solution and includes 30 mL of Qdot® incubation buffer.

Click-iT™ SDP Ester sDIBO Alkyne (Invitrogen™)

Click-iT SDP Ester sDIBO Alkyne reacts with azides via a copper-free Click chemistry reaction to produce SDP Ester (amine-reactive) bioconjugates. sDIBO alkynes are improved versions of our original DIBO cyclooctynes, yielding conjugates that are less “sticky” and give lower signal background in biological samples. Copper-free Click bio-conjugation reactions are ideal for surface labeling of live cells and also minimize damage to enzymes and fluorescent proteins like GFP or R-PE. Macromolecules that have been azide-modified enzymatically, chemically, or metabolically can be now be labeled easily, yielding more soluble bioconjugates with improved biological labeling utility.

• More soluble than DIBO cyclooctynes leading to more soluble conjugates
• Minimal background potential in cells and tissues compared to original DIBO cycloctynes

Zenon™ R-Phycoerythrin Rabbit IgG Labeling Kit (Invitrogen™)

Zenon labeling technology provides a fast, versatile and reliable method for producing antibody conjugates, even with very small (submicrogram) amounts of starting material. Antibody conjugates formed using Zenon technology may be used to stain cells in any protocol where a directly labeled primary antibody is suitable, including flow cytometry, imaging, high throughput and other applications. Moreover, this technology simplifies applications that previously were time consuming or not practical, such as the use of multiple mouse-derived antibodies in the same staining protocol.

View a selection guide for all Zenon™ antibody labeling kits and other antibody labeling products.

Zenon™ Horseradish Peroxidase Mouse IgG1 Labeling Kit (Invitrogen™)

Zenon® labeling technology provides a fast, versatile, and reliable method for adding a fluorescent label to an antibody. You need only a small amount of starting material, and the method is optimized for efficient labeling of antibodies in serum, ascites fluid, or hybridoma suspensions. Antibody conjugates formed using Zenon® technology may be used in any protocol where a directly labeled primary antibody is suitable, including flow cytometry, imaging, and high-throughput applications. This exclusive Molecular Probes® Zenon® labeling technology greatly simplifies the use of multiple mouse-derived antibodies in the same staining protocol.

Important Features of Zenon® Labeling Technology:

• Labeled antibodies typically ready to use in 10 minutes
• Requires only 1–20 μg primary antibody
• Simple, no purification required
• Flexible–over 24 fluorophores plus biotin, HRP, alkaline phosphatase, and TSA to choose from
• Multiplex with other mouse monoclonal antibodies simultaneously


Save Time and Antibody
Each kit comes with affinity-purified monovalent Fab fragment of a goat anti-Fc antibody (or, in the case of the Zenon® Goat IgG Labeling Kits, a rabbit anti-Fc antibody) that has been conjugated to one of our premier Alexa Fluor® dyes or to Pacific Blue™, Pacific Orange™, fluorescein, or Texas Red®-X dyes, biotin R-phycoerythrin (R-PE), allophycocyanin (APC), HRP, or alkaline phosphatase.

Formation of the Fab–antibody complex with the Zenon® Antibody Labeling Kits is extremely fast (5 min for complex, 5 min for blocking step). And Zenon® labeling is a reliable and reproducible method, even with as low 0.4 μg in 2 μL of primary antibody. There is minimal waste of expensive or difficult-to-obtain antibodies when using the Zenon® Antibody Labeling Kits.

Preserve Primary Antibody Function and Affinities
Reactive dye labeling of primary antibodies can have unpredictable and undesirable outcomes. Among these are reduced binding affinities by label addition in the binding pocket. Zenon® antibody labeling approach, targeted to the Fc tail, avoids this concern.

Moreover the Zenon® dye- and enzyme-labeled Fab fragments have been affinity purified during their preparation to help ensure their high affinity and selectivity for the Fc portion of the corresponding primary antibody. The procedure for chemical labeling of the Fab fragments protects the Fc-binding site, resulting in more active labeling reagents.

Many Fluorophore and Enzyme Labels Available
Zenon® immunolabeling technology makes it very easy to change fluorescent color combinations or detection methodologies by simply using a different dye- or enzyme-labeled Fab fragment from our extensive selection of over 100 Zenon® Antibody Labeling Kits. If larger quantities or covalent attachment of the label is desired, see Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices.

Zenon® Technology Simplifies the Use of Multiple Antibodies of the Same Isotype in the Same Protocol
The stability of the Zenon® complex is sufficient to allow sequential (or simultaneous) labeling of different targets in cells and tissues with multiple antibody complexes. Subsequent to staining, an aldehyde-based fixation step can permanently block the transfer of Zenon® labels between different primary antibodies and will preserve the staining pattern.

We’ll Make a Custom Antibody Conjugate for You
If you can’t find what you’re looking for in our stocked list, we’ll prepare a custom antibody conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

Related Links:

Zenon® Labeling Technology
Zenon® Technology: Versatile Reagents for Immunolabeling—Section 7.3

5-(Aminomethyl)Fluorescein, Hydrochloride (Invitrogen™)

The primary aliphatic amine of 5-(aminomethyl)fluorescein can be reversibly coupled to aldehydes and ketones to form a Schiff base - which can be reduced to a stable amine derivative by sodium borohydride (NaBH4) or sodium cyanoborohydride (NaCNH3) to form new probes. Carboxylic acids of proteins and other water-soluble biopolymers can be coupled to this molecule in aqueous solution using water-soluble carbodiimides such as EDAC (E2247).

QSY™ 7 Carboxylic Acid, Succinimidyl Ester (Invitrogen™)

QSY-7 succinimidyl ester is a nonfluorescent acceptor dye for preparation of peptide and oligonucleotide FRET probes via aliphatic amine modification.

Pacific Blue™ Succinimidyl Ester (Invitrogen™)

The amine-reactive Pacific Blue™ succinimidyl ester can be used to can be used to create blue-fluorescent bioconjugates with excitation/emission maxima ~410/455 nm that are excitable by the 405 nm spectral line of the blue diode (violet) laser.

View all Pacific Blue™ dye products..

View the Fluorophore Selection Guide.

EZ-Link™ NHS-PEG12-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link NHS-PEG12-Biotin is very long, pegylated biotinylation reagent for simple and efficient biotin labeling of antibodies, proteins and other primary amine-containing macromolecules.

Features of EZ-Link NHS-PEG12-Biotin:

Protein labeling—biotinylate antibodies or other proteins for detection or purification using streptavidin probes or resins
Amine-reactive—reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the amino-termini of polypeptides
Pegylated—spacer arm contains a hydrophilic, 12-unit, polyethylene glycol (PEG) group
Enhances solubility—pegylation imparts water solubility to the biotinylated molecule, helping to prevent aggregation of biotinylated antibodies stored in solution
Irreversible—forms permanent amide bonds; spacer arm cannot be cleaved
Very long reach—spacer arm (total length added to target) is 56 angstroms; this reduces steric hindrance when binding to avidin molecules

NHS-PEG12-Biotin is a very long (56.0Å), pegylated, water-soluble, NHS-ester biotinylation reagent to label amines and maximize solubility of antibodies and other proteins. The N-hydroxysuccinimide ester (NHS) group reacts specifically and efficiently with lysine and N-terminal amino groups at pH 7-9 to form stable amide bonds. The hydrophilic polyethylene glycol (PEG) spacer arm imparts water solubility that is transferred to the biotinylated molecule, thus reducing aggregation of labeled proteins stored in solution. The PEG spacer arm also gives the reagent a long and flexible connection to minimize steric hindrance for binding to avidin molecules.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.

N-Hydroxysulfosuccinimide (NHS) esters of biotin are the most popular type of biotinylation reagent. NHS-activated biotins react efficiently with primary amino groups (-NH2) in alkaline buffers to form stable amide bonds. Proteins (e.g., antibodies) typically have several primary amines that are available as targets for labeling, including the side chain of lysine (K) residues and the N-terminus of each polypeptide.

Varieties of biotin NHS-ester reagents differ in length, solubility, cell permeability and cleavability. Non-sulfonated NHS-biotins are cell permeable but must be dissolved in organic solvent such as DMSO or DMF. Sulfo-NHS biotins (and those with pegylated spacers) are directly water soluble but not membrane permeable. Varieties containing disulfide bonds can be cleaved using reducing agents, enabling the biotin group to be disconnected from the labeled protein.

pHrodo™ Red, succinimidyl ester (pHrodo™ Red, SE) (Invitrogen™)

New pH-sensitive pHrodo™ Red dye conjugates give faster and more accurate results than any other phagocytosis assay

pHrodo™ Red dye conjugates are non-fluorescent outside the cell, but fluoresce brightly red in phagosomes

Get faster staining and more accurate results - without the need for wash steps or quencher dye


• Specific detection of phagocytosis and endocytosis
• Reduced signal variability and improved timing in sensitive experiments
• Multiplex with green dyes such as GFP, Fluo-4, or calcein


The fluorescence of the novel pHrodo™ Red dye dramatically increases as pH decreases from neutral to the acidic, making it an ideal tool to study phagocytosis and its regulation by drugs and/or environmental factors. The lack of fluorescence outside the cell eliminates the need for wash steps and quencher dyes.

Use the ready-made pHrodo™ Red E.coli BioParticles® conjugates in imaging or flow applications, or pHrodo™ Red SE, the activated succinimidyl ester, for labeling microorganisms or proteins of your choice.

Pierce™ Premium Grade Sulfo-NHS-LC-Biotin (Thermo Scientific™)

Thermo Scientific Pierce Premium Grade Sulfo-NHS-LC-Biotin is our highest quality formulation of this popular amine-reactive biotinylation reagent, specially characterized for applications where product integrity and risk minimization are paramount.

Features of Premium Grade Sulfo-NHS-LC-Biotin:

Sulfo-NHS-LC-Biotin—popular amine-reactive biotinylation reagent for antibody labeling
High quality—identity and purity confirmed by several tests, including quantitative NMR
Product integrity—enhanced level of testing and characterization compared to standard grade
Lot retention—ample supply of past lots retained to ensure future process testing
Change management—Change Control Notification (CCN) service
Consistent manufacture—batch-specific manufacturing documentation review

Compared to the standard grade product, Premium Grade Sulfo-NHS-LC-Biotin provides more clearly defined quality and product support by including (a) increased analytical testing and product characterization, (b) greater batch-specific information and quality assurance review, (c) extensive lot sample retention and (d) change control notification. Sulfo-NHS-LC-Biotin is an intermediate-length, water-soluble biotinylation reagent for labeling antibodies, proteins and other molecules that have primary amines. Specific labeling of cell surface proteins is another common application for these uniquely water-soluble and membrane impermeable reagents.

Related Products
EZ-Link™ Sulfo-NHS-LC-Biotin

EZ-Link™ Sulfo NHS-LC-LC-Biotin, No-Weigh™ Format (Thermo Scientific™)

Thermo Scientific EZ-Link Sulfo-NHS-LC-LC-Biotin enables simple and efficient biotin labeling of antibodies, proteins, and any other primary amine–containing macromolecules. Specific labeling of cell surface proteins is another common application for these uniquely water-soluble and membrane impermeable reagents.

Thermo Scientific No-Weigh products are specialty reagents provided in a pre-aliquoted format. The pre-weighed packaging prevents the loss of reagent reactivity and contamination over time by eliminating the repetitive opening and closing of the vial. The format enables use of a fresh vial of reagent each time, eliminating the hassle of weighing small amounts of reagents and reducing concerns over reagent stability.

Features of EZ-Link Sulfo-NHS-LC-LC-Biotin:

Protein labeling—biotinylate antibodies to facilitate immobilization, purification or detection using streptavidin resins or probes
Cell surface labeling—biotinylates only surface proteins of whole cells because the negatively charged reagent does not permeate cell membranes
Amine-reactive—reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the amino-termini of polypeptides
Soluble—charged sulfo-NHS group increases reagent water solubility compared to ordinary NHS-ester compounds
Irreversible—forms permanent amide bonds; spacer arm cannot be cleaved
Doubly long —spacer arm (total length added to target) is 22.4 angstroms; this extended arm helps to minimize steric hindrance for biotin binding

Sulfo-NHS-LC-LC-Biotin is the longest of three very similar EZ-Link Reagents that are water-soluble, non-cleavable, and enable simple and efficient biotinylation of antibodies, proteins and any other primary amine-containing macromolecules in solution. Specific labeling of cell surface proteins is another common application for these uniquely water-soluble and membrane impermeable reagents. Differing only in their spacer arm lengths, the three Sulfo-NHS-ester reagents offer the possibility of optimizing labeling and detection experiments where steric hindrance of biotin binding is an important factor.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency, and performance for the intended research applications.

N-Hydroxysulfosuccinimide (NHS) esters of biotin are the most popular type of biotinylation reagent. NHS-activated biotins react efficiently with primary amino groups (-NH2) in alkaline buffers to form stable amide bonds. Proteins (e.g., antibodies) typically have several primary amines that are available as targets for labeling, including the side chain of lysine (K) residues and the N-terminus of each polypeptide.

Varieties of biotin NHS-ester reagents differ in length, solubility, cell permeability and cleavability. Non-sulfonated NHS-biotins are cell permeable but must be dissolved in organic solvent such as DMSO or DMF. Sulfo-NHS biotins (and those with pegylated spacers) are directly water soluble but not membrane permeable. Varieties containing disulfide bonds can be cleaved using reducing agents, enabling the biotin group to be disconnected from the labeled protein.

BODIPY™ FL-X NHS Ester (Succinimidyl Ester) (Invitrogen™)

BODIPY® FL-X dye is bright, green fluorescent dye with similar excitation and emission to fluorescein (FITC) or Alexa Fluor® 488 dye. It has a high extinction coefficient and fluorescence quantum yield and is relatively insensitive to solvent polarity and pH change. In contrast to the highly water soluble fluorophores Alexa Fluor® 488 dye and fluorescein (FITC), BODIPY® dyes have unique hydrophobic properties ideal for staining lipids, membranes, and other lipophilic compounds. BODIPY® FL-X dye has a relatively long excited-state lifetime (typically 5 nanoseconds or longer), which is useful for fluorescence polarization-based assays and a large two-photon cross-section for multiphoton excitation. In addition to reactive dye formulations, we offer BODIPY® FL-X dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

The NHS ester (or succinimidyl ester) of BODIPY® FL-X is the most popular tool for conjugating the dye to a protein or antibody. NHS esters can be used to label the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting BODIPY® FL-X conjugates exhibit bright fluorescence, narrow emission bandwidths, and relatively long excited-state lifetimes, which can be useful for fluorescence polarization assays and two-photon excitation (TPE) microscopy.

This reactive dye contains a seven-atom aminohexanoyl ("X") spacer between the fluorophore and the NHS ester group. This spacer helps to separate the fluorophore from its point of attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated.

Detailed information about this BODIPY® FL-X NHS ester:

Fluorophore label: BODIPY® FL-X dye
Reactive group: NHS ester (succinimidyl ester)
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 504/510 nm
Extinction coefficient: 85,000 cm-1M-1
Molecular weight: 502.32

Typical Conjugation Reaction
Amine-reactive reagents can be conjugated with virtually any protein or peptide; the provided protocol is optimized for IgG antibodies. The reaction can be scaled for any amount of protein, but the concentration of the protein should be at least 2 mg/mL for optimal results. We recommend trying three different degrees of labeling, using three different molar ratios of the reactive reagent to protein.

The BODIPY® NHS ester is typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO), and the reaction is carried out in 0.1-0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature for 1 hour. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, you may achieve more selective labeling of the amine terminus using a buffer closer to neutral pH.

Conjugate Purification
Labeled antibodies are typically separated from free BODIPY® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration medium with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

PyMPO, SE, 1-(3-(Succinimidyloxycarbonyl)Benzyl)-4-(5-(4-Methoxyphenyl)Oxazol-2-yl)Pyridinium Bromide (Invitrogen™)

The amine-reactive PyMPO succinimidyl ester can be used to create bioconjugates with this environment-sensitive fluorophore.

DyLight™ 488, Free Acid (Thermo Scientific™)

Thermo Scientific DyLight Fluor Free Acids are non-activated forms of selected DyLight Fluorescent Dyes for use as controls and calibration factors in fluorescence imaging applications. DyLight 488 has excitation and emission peaks at 493 and 518 nm, respectively (±4 nm, in PBS).

These non-activated fluorescent dyes contain the native carboxyl group of the standard dye molecule.

General DyLight Fluorophore Highlights:

Bright fluorescence—intense emission provides superior sensitivity and requires less conjugate
Narrow emission spectra—negligible bleed-through between fluorophore channels enables multi-color detection
Excellent photostability—exceptional resistance to photobleaching enables fluorescence imaging under the most demanding conditions (e.g., structured illumination, 4Pi microscopy)
Buffer stability—conjugated fluorophores are completely stable at pH 4-9
Instrument compatibility—excitation and emission spectra correspond with filter sets and laser settings of all popular fluorescence instrumentation

Related Products
DyLight™ 747, Free Acid
DyLight™ 800, Free Acid

Qdot™ 655 ITK™ Amino (PEG) Quantum Dots (Invitrogen™)

Qdot® 655 ITK™ amino (PEG) quantum dots are the ideal starting material for preparing custom conjugates of ultrabright and photostable fluorescently labeled proteins or other biopolymers. These probes are functionalized with amine-derivatized PEG, which prevents non-specific interactions and provides a convenient handle for conjugation. The amino quantum dots react efficiently with isothiocyanates and succinimidyl esters, or with native carboxylic acids using water-soluble carbodiimides such as EDC. Such derivatives may be used for various labeling and tracking applications that require ultrabright and stable fluorescence. Our Qdot® ITK™ amino quantum dots are provided as 8 µM solutions and are available in 8 colors of Qdot® probes.

Important Features of Qdot® ITK™ Amino Quantum Dots:
• Qdot® 655 ITK™ amino quantum dot has emission maxima of ~655 nm
• Extremely photostable and bright fluorescence
• Efficiently excited with single-line excitation sources
• Narrow emission, large stokes shift
• Available in multiple colors
• Ideal for various labeling and tracking applications


Properties of Qdot® Nanocrystals
Qdot® probes are ideal for imaging and labeling applications that require bright fluorescent signals and/or real-time tracking. Unique among fluorescent reagents, all nine available colors of Qdot® probes can be simultaneously excited with a single (UV to blue-green) light source. This property makes these reagents excellent for economical and user-friendly multiplexing applications. Qdot® labels are based on semiconductor nanotechnology and are similar in scale to moderately sized proteins.

About the Innovator’s Tool Kit Qdot® ITK™ Reagents
These Qdot® ITK™ probes are ideal for researchers who wish to prepare specific (non-stocked) conjugates for their applications and need customizable conjugation functionality.

Other Forms of Qdot® Nanocrystals are Available
In addition to the amine-derivatized form, we offer Qdot® ITK™ quantum dots with carboxyl and aliphatic hydrocarbon modifications. We’ve also developed a wide range of Qdot® nanocrystals conjugates and labeling kits. Investigate the properties of Qdot® nanocrystals or read the Molecular Probes® Handbook Section 6.6—Qdot® Nanocrystals to find out more.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

DyLight™ 550 NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight 550 Amine-Reactive Dye is an NHS ester-activated derivative of high-performance DyLight 550 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods.

DyLight 550 provides vibrant orange-to-red fluorescence with better performance than other rhodamine derivatives, including Alexa Fluor™ 555, TRITC, and Cy3™ dye for fluorescent applications. The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates. DyLight 550 Amine-Reactive Dye is also available as part of two antibody labeling kit sizes.

Features of DyLight 550 NHS Ester:

High performance—DyLight 550 shows brighter fluorescence than Alexa Fluor 555, TRITC, and Cy3 dye
Specific—NHS ester-activated dye labels proteins and other molecules at primary amines (-NH2)
Optimized procedure—following the standard protocol results in antibodies with excellent dye:protein ratios and recovery rates for optimum activity and fluorescence labeling

Applications:
• Primary antibody labeling for immunofluorescence microscopy, immunohistochemistry (IHC), Western blotting, or ELISA assay
• Target protein labeling for in vitro and in vivo fluorescent detection strategies

DyLight 550 Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

Typical labeling reactions require DyLight 550 Amine-Reactive Dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to specific amounts of the labeling reagent. This feature allows DyLight 550 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

We also offer Standard and Microscale DyLight 550 Antibody Labeling Kits for fast and efficient fluorescent labeling of antibodies for use in fluorescence methods. The standard size kit contains all necessary components to perform three separate labeling reactions using 1 mg of IgG or similar quantities of other proteins. The microscale kit contains all of the necessary components to perform five separate labeling reactions using 100 µg of IgG. Both kit sizes include the Amine-Reactive DyLight 550 NHS-ester in convenient single-use vials as well as purification resin and spin columns for the preparation of ready-to-use conjugate.

Related Products
DyLight™ 550 Antibody Labeling Kit
DyLight™ 550 Microscale Antibody Labeling Kit

NHS-Fluorescein (5/6-carboxyfluorescein succinimidyl ester), mixed isomer (Thermo Scientific™)

NHS-Fluorescein is an amine-reactive derivative of fluorescein dye that has wide-ranging applications as a label for antibodies and other probes, for use in fluorescence microscopy, flow cytometry and immunofluorescence-based assays such as Western blotting and ELISA. The NHS-ester of fluorescein efficiently labels antibodies and other purified proteins at primary amines (lysine side chains)

Properties of NHS-Fluorescein:

• Alternative name: 5/6-FAM SE
• Chemical name: 5/6-carboxyfluorescein succinimidyl ester
• Molecular weight: 473.4
• Excitation source: 488 nm spectral line, argon-ion laser
• Excitation wavelength: 494 nm
• Emission wavelength: 518 nm
• Extinction coefficient: > 70,000 M-1cm-1
• CAS #: 117548-22-8
• Purity: > 90% by HPLC
• Solubility: Soluble in DMF or DMSO
• Reactive groups: NHS ester, reacts with primary amines at pH 7.0 to 9.0

NHS-fluorescein is activated with the N-hydroxy-succinimidyl-ester (NHS ester) functional group. Compared to FITC, the NHS-ester deriviative has greater specificity toward primary amines in the presence of other nucleophiles and results in a more stable linkage following labeling. Pierce Amine-reactive Fluorescein Dyes are mixtures of isomers with reactive groups attached at the 5- and 6-positions of the bottom ring. The properties of these isomers are indistinguishable in terms of excitation and emission spectra, and for protein applications there is no need to isolate a specific isomer.

Applications
• Label antibodies for use as immunofluorescent probes
• Label oligonucleotides for hybridization probes
• Detect proteins in gels and on Western blots

Related Products
FITC (5/6-fluorescein isothiocyanate), mixed isomer
Pierce™ NHS-Fluorescein Antibody Labeling Kit
Pierce™ FITC Antibody Labeling Kit

DyLight™ 550-2xPEG NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight 550-2xPEG Amine-Reactive Dye is a derivative of our high-performance DyLight 550 Dye that can be used to fluorescently label antibodies and other proteins.

The DyLight 550-2xPEG dye contains 2 polyethylene glycol (PEG) chains that are non-cytotoxic, enhance fluorescence, and reduce nonspecific binding of conjugates made with them. Conjugates made with DyLight 550-2xPEG Dye can be used as molecular probes for cellular imaging, flow cytometry, and other fluorescence detection methods. The PEG chains also improve solubility of the dyes and labeled molecules in aqueous solution, aid in cell permeability, and improve tissue retention.

Features of DyLight 550-2xPEG NHS Ester:

High fluorescence intensity—significantly brighter fluorescence than Alexa Fluor™ 555
PEGylated—improves solubility in aqueous solution and aids in cell permeability

Applications:
• Fluorescence microscopy
In vivo or ex vivo imaging
• Cell-based assays
• Flow cytometry/fluorescence-activated cell sorting (FACS)

DyLight 550-2xPEG Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

Typical labeling reactions require the dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to the labeling reagent.

CellTracker™ Blue CMHC Dye (Invitrogen™)

CellTracker™ Blue CMHC (4-chloromethyl-7-hydroxycoumarin) is a fluorescent dye well suited for monitoring cell movement or location. This dye is well retained, allowing for multigenerational tracking of cellular movements. And the blue excitation/emission spectra are ideal for multiplexing with green and red fluorescent dyes and proteins.

Need a different emission spectrum or longer tracking? View our other mammalian cell tracking products.

• Easy to use—remove medium, add dye, incubate 30 minutes, and image cells
• Fluorescent signal retention of >72 hours (typically three to six generations)
• Blue excitation/emission spectra (372/470 nm maxima) ideal for multiplexing
• Low cytotoxicity—does not affect viability or proliferation

CellTracker™ Blue CMHC fluorescent dye has been designed to freely pass through cell membranes into cells, where it is transformed into cell membrane-impermeant reaction products. CellTracker™ Blue CMHC dye is retained in living cells through several generations. The dye is transferred to daughter cells, but not adjacent cells in a population. CellTracker™ Blue CMHC dye is designed to display fluorescence for at least 72 hours, and the dye exhibits ideal tracking properties: it is stable, nontoxic at working concentrations, well retained in cells, and brightly fluorescent at physiological pH. Additionally, the excitation and emission spectra of CellTracker™ Blue CMHC dye are well separated from GFP (green fluorescent protein) and RFP (red fluorescent protein) spectra allowing for multiplexing.

Click-IT™ GlcNAz Metabolic Glycoprotein Labeling Reagent (tetraacetylated N-Azidoacetylglucosamine) (Invitrogen™)

The Click-iT® GlcNAz metabolic glycoprotein labeling reagent provides the first part of a simple and robust two-step technique to identify and characterize intracellular O-GlcNAc glycoproteins. In step one, cultured cells are incubated with the azide-modified glucosamine (GlcNAc). The azido-sugar is incorporated into intracellular O-GlcNAc-containing glycoproteins through the permissive nature of the oligosaccharide biosynthesis pathway. In step two, via the chemoselective ligation or click reaction between an azide and an alkyne, the azido-labeled glycoproteins can then detected with a Click-iT® Glycoprotein Detection kit for gels (TAMRA or Dapoxyl® alkyne) or Western blots (biotin alkyne). The Click-iT® glycoprotein products are compatible with LC-MS⁄MS and Multiplexed Proteomics™ technologies for in-depth analyses of the glycoproteome.

pHrodo™ iFL Green STP Ester (amine-reactive) (Invitrogen™)

The amine-reactive pH-sensitive pHrodo iFL Green STP Ester dye is an improved version of pHrodo Green dye optimized for the creation of bioconjugates to be used in the study of antibody internalization, endocytosis, and phagocytosis. pHrodo iFL Green dye dramatically increases fluorescence as the pH of its surroundings become more acidic. It is more soluble than the original pHrodo Green dye, making it more useful for the labeling of antibodies that may otherwise precipitate out of solution during conjugation.

• Use pH-sensitive pHrodo iFL Green STP Ester to make pH-sensitive bioconjugates of your choice
• Get faster, more accurate results than with any other endocytosis or phagocytosis assay—no need for wash steps or quenchers
• Multiplex with red fluorescent markers such as RFP, pHrodo iFL Red STP, and many others

The increase in fluorescence of pHrodo iFL Green dye as the pH changes from basic to acidic correlates with the acidification of intracellular vesicles, making it a valuable tool for the study of endocytosis or phagocytosis and their regulation by environmental factors, drugs, or pathogens. The spectral properties of pHrodo iFL Green dye make it useful for multi-color experiments. pHrodo iFL Green dye can be detected using most standard green-fluorescent filter sets and has been validated for use in a variety of applications, including flow cytometry, fluorescent microscopy, and high content screening (HCS). The lack of fluorescence of pHrodo iFL Green dye in a typical extracellular environment eliminates the need for wash steps or quencher dyes in the experimental workflow.

pHrodo iFL Green STP Ester is an amine-reactive dye that can be used to make pHrodo iFL Green STP bioconjugates in aqueous buffer. The STP ester will react with primary amines on a protein, cell, or virus to create a stable conjugate that can be used in live cell assays or stored for later use.

DyLight™ 680 NHS-Ester (Thermo Scientific™)

Thermo Scientific DyLight 680 Amine-Reactive Dye is an NHS ester-activated derivative of high-performance DyLight 680 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods.

DyLight 680 produces near-infrared (IR) fluorescence that replaces other near-IR dyes, including Cy5.5™ dye and Alexa Fluor™ 680, and is ideal for multi-color applications. The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates. DyLight 680 Amine-Reactive Dye is also available as part of two antibody labeling kit sizes.

Features of DyLight 680 NHS-Ester:

High performance—DyLight 680 fluoresces brighter than Alexa Fluor 680 and Cy5.5 dye
Specific—NHS ester-activated dye labels proteins and other molecules at primary amines (-NH2)
Optimized procedure—following the standard protocol results in antibodies with excellent dye:protein ratios and recovery rates for optimum activity and fluorescence labeling

Applications:
• Primary antibody labeling for immunofluorescence microscopy, immunohistochemistry (IHC), Western blotting, or ELISA assay
• Target protein labeling for in vitro and in vivo fluorescent detection strategies

DyLight 680 Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

Typical labeling reactions require the dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to specific amounts of the labeling reagent. This feature allows DyLight 680 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

We also offer Standard and Microscale DyLight 680 Antibody Labeling Kits for fast and efficient fluorescent labeling of antibodies for use in fluorescence methods.The standard size kit contains all necessary components to perform three separate labeling reactions using 1 mg of IgG or similar quantities of other proteins. The microscale kit contains all of the necessary components to perform five separate labeling reactions using 100 µg of IgG. Both kit sizes include the Amine-Reactive DyLight 680 NHS-ester in convenient single-use vials as well as purification resin and spin columns for the preparation of ready-to-use conjugate.

Related Products
DyLight™ 680 Antibody Labeling Kit
DyLight™ 680 Microscale Antibody Labeling Kit

Tetramethylrhodamine (TAMRA) Alkyne (5-Carboxytetramethylrhodamine, Propargylamide), 5-isomer (Invitrogen™)

The red-fluorescent tetramethylrhodamine (TAMRA) alkyne can be reacted with azides via a copper-catalyzed click reaction. Click chemistry describes a class of chemical reactions that use bio-orthogonal or biologically unique moities to label and detect a molecule of interest using a two-step procedure. The two-step reaction procedure involves a copper-catalyzed triazole formation of an azide and an alkyne. Click reactions have several characteristics: the reaction between the detection moieties is efficient; no extreme temperatures or solvents are required; the reaction product is stable; the components of the reaction are bioinert; and perhaps most importantly, no side reactions occur – the label and detection tags react selectively and specifically with one another. Unlike traditional chemical reactions utilizing succinimidyl esters or maleimides that target amines and sulfhydryls – functional groups that are not unique – click chemistry-labeled molecules can be applied to complex biological samples and be detected with unprecedented sensitivity due to extremely low background.

Pacific Blue™ Antibody Labeling Kit (Invitrogen™)

Molecular Probes™ Antibody Labeling Kits provide a convenient means to label small amounts of antibodies with the violet light-excitable Pacific Orange™ or Pacific Blue™ dyes. This kit is optimized for labeling 100 µg of antibody per reaction. Comparably small amounts of other proteins (>40 kDa) can also be labeled.

View a selection guide for all Antibody Labeling Kits.

View the Fluorophore Selection Guide.

The kit contains everything you need to perform five separate labeling reactions as well as to purify the resulting conjugates. Conjugates are ideal for multiple applications, including flow cytometry, fluorescent microscopy, immunohistochemistry, primary detection, ELISAs, immunocytochemistry, indirect FISH, and more.

Important features of antibody labeling kits:
• Pacific Blue™ has an excitation and emission maximum of 410/455 nm
• Labeled proteins typically ready to use in 90 min (~15 min hands-on time)
• Useful for labeling 100 µg of protein
• Optimized for small-scale labeling of any protein >40 kDa
• Purified using convenient spin filters
• Stabilizing proteins must be removed from the sample before labeling
• Includes detailed instructions for determining degree of labeling (DOL)


Better results and workflows with primary labeled antibodies
A primary antibody directly labeled with a fluorophore often produces lower background fluorescence and less nonspecific binding. Further, multiple primary antibodies of the same isotype or derived from the same species can easily be used in the same experiment if they are directly labeled with compatible fluorophores.

Learn more about protein and antibody labeling
We offer a wide selection of Molecular Probes™ antibody and protein labeling kits to fit your starting material and your experimental setup. See Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices. To learn more about our various kits read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in the Molecular Probes™ Handbook.

We'll make a custom antibody conjugate for you
If you can't find what you're looking for in our stocked list, we'll prepare a custom antibody conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

Qtracker™ 655 Vascular Labels (Invitrogen™)

Qtracker® non-targeted quantum dots are designed to be injected into the tail vein of mice for the study of vascular structure using small animal in vivo imaging (SAIVI) techniques. These nanocrystals exhibit intense fluorescence with red-shifted emission for increased tissue penetration, and have a PEG surface coating specially developed to minimize nonspecific interactions and reduce immune response by the tissue. Because the PEG surface coating does not contain reactive functional groups, the Qtracker® non-targeted quantum dots are retained in circulation longer and can be imaged for up to 3 hours with a single injection or for longer periods of time with additional injections.

Need a different emission spectrum or longer tracking? View our other mammalian cell tracking products.

Key Attributes:

Qtracker® 655 label has Ex/Em (405-615/655) nm
Designed for small animal in vivo imaging
Introduced via tail vein injection, can be imaged for up to 3 hours after injection
Available in four colors—565 nm, 655 nm, 705 nm, or 800 nm emission

Read more about SAIVI and about applications for Qdot® nanocrystals.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Alexa Fluor™ 633 NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor® 633 is a bright and photostable far-red dye with excitation ideally suited to the 633 nm laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 633 dye is water soluble and pH-insensitive from pH 4 to pH 10. Fluorescence of this long-wavelength Alexa Fluor® dye is not visible to the human eye but is readily detected by most imaging systems. In addition to reactive dye formulations, we offer Alexa Fluor® 633 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection.The NHS ester (or succinimidyl ester) of Alexa Fluor® 633 is the most popular tool for conjugating this dye to a protein or antibody. NHS esters can be used to label to the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting Alexa Fluor® conjugate will exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores.

Detailed information about this AlexaFluor® NHS ester:

Fluorophore label: Alexa Fluor® 633 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 621/639 nm
Extinction coefficient: 159,000 cm-1M-1
Spectrally similar dyes: Cy5®
Molecular weight: ~1200

Typical Conjugation Reaction
You can conjugate amine-reactive reagents with virtually any protein or peptide (the provided protocol is optimized for IgG antibodies). You can scale the reaction for any amount of protein, but the concentration of the protein should be at least 2 mg/mL for optimal results. We recommend trying three different degrees of labeling, using three different molar ratios of the reactive reagent to protein.

The Alexa Fluor® NHS ester is typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO) (D12345), and the reaction is carried out in 0.1–0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature for 1 hour. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, you may achieve more selective labeling of the amine terminus using a buffer closer to neutral pH.

Conjugate Purification
Labeled antibodies are typically separated from free Alexa Fluor® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

Biotin Alkyne (PEG4 carboxamide-Propargyl Biotin) (Invitrogen™)

The hapten, biotin azide is reactive with terminal via a copper-catalyzed click reaction. Biotin can be subsequently detected with streptavidin, avidin or NeutrAvidin® biotin-binding protein.

EZ-Link™ NHS-LC-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link NHS-LC-Biotin is a long-chain, NHS-ester activated biotinylation reagent for labeling primary amines (e.g., protein lysines), whose membrane permeability enables it to be used for general intracellular labeling.

Features of EZ-Link NHS-LC-Biotin:

Protein labeling—biotinylate antibodies or other proteins for detection or purification using streptavidin probes or resins
Membrane-permeable—can be used to label inside cells (intracellular)
Amine-reactive—reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the amino-termini of polypeptides
Irreversible—forms permanent amide bonds; spacer arm cannot be cleaved
Solubility—must be dissolved in DMSO or DMF before further dilution in aqueous buffers
Medium length—spacer arm (total length added to target) is 22.4 angstroms; it consists of the biotin valeric acid group extended by a 6-atom chain

NHS-LC-Biotin is succinimidyl-6-(biotinamido)hexanoate. It is one of three similar EZ-Link NHS-Biotin Reagents that enable simple and efficient biotinylation of antibodies, proteins and any other primary amine-containing biomolecules in solution. Differing only in their spacer arm lengths, the three NHS-ester reagents offer researchers the possibility of optimizing labeling and detection experiments where steric hindrance of biotin binding is an important factor. Because they are uncharged and contain simple alkyl-chain spacer arms, these biotin compounds are membrane-permeable and useful for intracellular labeling.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.

N-Hydroxysulfosuccinimide (NHS) esters of biotin are the most popular type of biotinylation reagent. NHS-activated biotins react efficiently with primary amino groups (-NH2) in alkaline buffers to form stable amide bonds. Proteins (e.g., antibodies) typically have several primary amines that are available as targets for labeling, including the side chain of lysine (K) residues and the N-terminus of each polypeptide.

Varieties of biotin NHS-ester reagents differ in length, solubility, cell permeability and cleavability. Non-sulfonated NHS-biotins are cell permeable but must be dissolved in organic solvent such as DMSO or DMF. Sulfo-NHS biotins (and those with pegylated spacers) are directly water soluble but not membrane permeable. Varieties containing disulfide bonds can be cleaved using reducing agents, enabling the biotin group to be disconnected from the labeled protein.

EZ-Link™ Pentylamine-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link Pentylamine-Biotin is biotin that has been conjugated to pentylenediamine to add a short, amine-terminated spacer to its valeric acid group, providing a simple amine-modified compound for different applications.

Features of EZ-Link Pentylamine-Biotin:

Biotinylation—label molecules and surfaces for assay or affinity purification methods involving avidin or streptavidin probes and resins
Amine-activated—primary amine can be crosslinked to proteins and material surfaces using EDC and other crosslinkers
Medium length—spacer arm (total length added to target) is 18.9 angstroms, representing a 6-atom extension of the native biotin valeric acid (and its conversion to a primary amine)

Pentylamine Biotin is a compound formed by modification of the valeric acid side chain of biotin with pentane-1,5-diamine. The compound contains terminal primary amine (-NH2), which provides a functional handle for derivatization or conjugation to proteins, surfaces and other molecules. Carbodiimide (EDC) and NHS-ester crosslinker chemistries are most often utilized for covalent modifications involving amino-biotin molecules. The compound is also useful as an amine-alternative to native (carboxylate) biotin for assays and methods that require a biotin standard or blocking step.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.

Amino-biotin compounds can be conjugated to functional groups of proteins and other molecules in a variety of ways. The most common method is to crosslink the terminal primary amine to carboxyl groups using . Carboxyl groups (-COOH) occur in aspartate or glutamate residues and the carboxy-terminus of polypeptides. When activated with EDC (Part No. 22980), carboxylates react with amino (—NH2) groups to form amide bonds. Carboxylate molecules and surface materials can be pre-activated using EDC with Sulfo-NHS (Part No. 24510) for subsequent reaction to primary amines (see NHS-ester Chemistry).

Applications:
• Used for colorimetric assays for Factor XIII and cellular transglutaminase

SAIVI™ Rapid Antibody Labeling Kit, Alexa Fluor™ 750 (Invitrogen™)

The Alexa Fluor® 750 SAIVI™ Antibody Labeling Kit provides a convenient means to label antibodies with an optimal degree of labeling for in vivo imaging applications (DOL; ~2) over a 6-fold antibody concentration range with no adjustments in reaction volume, dye concentration, or antibody concentration necessary. Using this procedure, optimally labeled antibodies are ready for applications that require azide-free reagents, such as live-cell imaging or direct injection into animals.