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EZ-Link™ Sulfo NHS-SS Biotinylation Kit (Thermo Scientific™)

The Thermo Scientific EZ-Link Sulfo NHS-SS Biotinylation Kit contains sufficient reagents for 10 biotinylation reactions (e.g., 1–10 mg antibody per reaction). The EZ-Link Sulfo-NHS-SS-Biotin included in the kit is a water-soluble, NHS-ester biotinylation reagent whose spacer arm includes a cleavable disulfide bond for reversible labeling of proteins and cell surface primary amines.

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

Protein labeling – biotinylate antibodies to facilitate immobilization, purification or detection
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 lysine side-chains, or the amino-termini of polypeptides
Cleavable—disulfide bond in spacer arm allows the biotin label to be removed using reducing agents such as DTT; only a small sulfhydryl group remains attached to the molecule
Soluble – charged sulfo-NHS group increases reagent water solubility compared to ordinary NHS-ester compounds
Medium length—spacer arm (total length added to target) is 24.3 angstroms; it consists of the native biotin valeric acid group extended by a 7-atom chain

Sulfo-NHS-SS-Biotin is a thiol-cleavable amine-reactive biotinylation reagent that contains an extended spacer arm to reduce steric hindrances associated with avidin binding. This reagent is water-soluble, enabling biotinylation to be performed in the absence of organic solvents such as DMSO or DMF for applications that cannot tolerate solvents or are complicated by their inclusion. The compound is particularly useful for labeling and purifying cell surface proteins, because (a) its sulfonate group prevents it from permeating cell membranes and (b) its cleavable spacer arm enables initially biotinylated proteins to be released from streptavidin affinity columns.

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.

Related Products
EZ-Link™ Sulfo-NHS-SS-Biotin
EZ-Link™ Micro Sulfo-NHS-SS-Biotinylation Kit

Texas Red™ Sulfonyl Chloride, mixed isomers (Invitrogen™)

The amine-reactive Texas Red® sulfonyl chloride can be used to can be used to create bright red-fluorescent bioconjugates with excitation/emission maxima ~595/615 nm.

Tetramethylrhodamine-5-Iodoacetamide Dihydroiodide (5-TMRIA), single isomer (Invitrogen™)

The thiol-reactive tetramethylrhodamine-5-iodoacetamide dihydroiodide (5-TMRIA) can be used to can be used to create bright orange-red-fluorescent bioconjugates with excitation/emission maxima ~555/580.

Zenon™ Tricolor Mouse IgG2b Labeling Kit #1 (for Green, Orange and Deep Red Fluorescence Imaging) (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. Zenon® Tricolor Labeling Kits contain sufficient materials for 10 labeling reactions of each of three different fluorescent colors.

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

DyLight™ 830-B2 NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight near-infrared specialty dyes, comparable to Alexa Fluor and IRDye NIR dyes, can be used to label antibodies, peptides, and other proteins at primary amines. DyLight 830-B2 dye has a structure based on the benzopyrillium core, with 2 sulfonates. It has excitation and emission peaks at 844 and 875 nm, respectively (in ethanol).

General characteristics of DyLight near-infrared emitting specialty dyes:

Large selection—the largest family of dyes available for NIR fluorescence applications
NHS ester reactive group—allows immediate labeling of antibodies, proteins, peptides and other amine-containing molecules through amide bond formation
Broad spectrum of water solubilities—choose from hydrophilic to hydrophobic dyes to optimize the right dye label for the best performance in a given application
NIR dyes avoid background interference—DyLight NIR Dyes avoid fluorescence interference or quenching effects from biomolecules present in samples
Excellent signal penetration through cells and tissues—DyLight NIR Dyes provide the optimal window for excitation and emission for in vivo imaging applications

DyLight NIR Dyes are a family of labeling agents that can be used for bright fluorescence detection in cell-based imaging or in vivo imaging applications. NIR dyes can be selected based upon their characteristic excitation and emission properties or relative hydrophilicity and hydrophobicity attributes. Dyes that contain a greater number of negatively charged sulfonates generally will have greater water solubility than dyes with fewer sulfonates. More hydrophobic dyes often provide better cell penetrating ability in vivo, while more hydrophilic dyes have less nonspecific binding potential. Each dye contains an amine-reactive NHS ester for simple modification of antibodies, proteins, peptides or other biomolecules through amide bond formation. NIR dyes are best for imaging through tissues and away from indigenous fluorescent biomolecule interference or quenching. DyLight Near Infrared Dyes represent the largest selection of fluorescent labels that are commercially available.

Criteria to consider when choosing a DyLight NIR Specialty Dye
• Excitation and emission wavelengths—choose the best dye to match the excitation and emission capabilities of your instrument
• Water solubility—choose a DyLight NIR Dye based on its relative hydrophilicity, which directly correlates to the number of negatively-charged sulfonates it has on its core structure. More hydrophilic dyes are best at maintaining water solubility of a labeled antibody and limiting the nonspecific binding of the conjugate. More hydrophobic dyes often are best at penetrating tissues and cell membranes in vivo, meaning that dyes with fewer sulfonates may work best for some applications.
• DyLight Dye selection—the broad selection of NIR dyes allows a number of candidate dyes to be tested in a given application for optimal performance.

Applications:
In vivo or ex vivo imaging
• Tumor imaging with labeled peptides
• NIR fluorescence (NIRF) imaging of labeled silica nanoparticles
• NIR in vitro imaging and characterization
• Determination of thermal stability
• Cytotoxicity assays
• Molecular imaging
• UV-VIS-NIR spectroscopy
• Fluorescence correlation spectroscopy
• MRI applications
• DNA sequencing
• Primer labeling for PCR
• 2-D gel electrophoresis
• Flow cytometry/fluorescence-activated cell sorting (FACS)
• Laser scanning confocal microscopy

Related Products
DyLight™ 780-B1 NHS Ester
DyLight™ 780-B2 NHS Ester
DyLight™ 780-B3 NHS Ester

Click-iT™ Plus OPP Alexa Fluor™ 647 Protein Synthesis Assay Kit (Invitrogen™)

The Click-iT® Plus OPP Alexa Fluor® 647 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® 647 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® 647 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 to produce low backgrounds and high detection sensitivities. Because of the mild reaction conditions, Click-iT® Plus assays detect protein translation events while enabling 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.

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

N-(Biotinoyl)-N'-(Iodoacetyl)Ethylenediamine (Invitrogen™)

The thiol-reactive biotin iodoacetamide reagent can be used to covalently attach biotin to thiol-containing proteins or thiolated nucleic acids.  The addition of a biotin residue enables the detection with avidin or streptavidin conjugates.  Electrophoretically separated thiol-containing proteins treated with biotin iodoacetamide have been detected in Western blots using an avidin–alkaline phosphatase conjugate.

Alexa Fluor™ 680 C2 Maleimide (Invitrogen™)

Alexa Fluor® 680 is a bright, near-infrared fluorescent dye with excitation ideally suited for the 633 nm laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 680 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 680 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® 680 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® 680 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® 680 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 684/714 nm
Extinction coefficient: 175,000 cm-1M-1
Spectrally similar dyes: Cy5.5, IRDye 680LT
Molecular weight: ~1000

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.

APEX™ Biotin-XX Antibody Labeling Kit (Invitrogen™)

The APEX® Antibody Labeling Kits are our best option for covalently attaching a fluorophore to small amounts of IgG antibody (~10–20 μg). It is ideal for the efficient labeling of antibodies in serum, ascites fluid, or hybridoma suspensions. Labeled antibodies are ready for use in imaging or flow cytometry applications in as little as 2.5 hours with very little hands on time.

Important Features of Alexa Fluor® APEX® Antibody Labeling Kits:

• Labeled antibodies typically ready to use in 2.5 hours (~15 minutes hands on time)
• Designed to label 10–20 μg of IgG
• Covalent attachment
• Compatible with contaminating proteins or stabilizers like BSA
• No columns needed; everything you need is supplied for 5 separate labelings
• Choose from Alexa Fluor® 488, 555, 568, 594, and 647 dyes, Oregon Green® 488 dye, Pacific Blue™ dye, and Biotin-XX.


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.

Contaminating Proteins or Protein Stabilizers Are Not a Problem
Many IgG antibodies are often available only in small quantities and packaged with stabilizing proteins, such as BSA, or other contaminants which can interfere with the amine-reactive labeling reagents. The APEX® Antibody Labeling Kits avoids this by utilizing a solid-phase labeling technique that captures the IgG antibody on the resin inside the APEX® antibody labeling tip. Contaminants are simply eluted through the tip, prior to applying the amine-reactive label.

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.

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

BODIPY™ 530/550 NHS Ester (Succinimidyl Ester) (Invitrogen™)

BODIPY® 530/550 dye is bright, orange fluorescent 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® 530/550 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® 530/550 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® 530/550 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® 530/550 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® 530/550 NHS ester:

Fluorophore label: BODIPY® 530/550 dye
Reactive group: NHS ester (succinimidyl ester)
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 534/551 nm
Extinction coefficient: 77,000 cm-1M-1
Molecular weight: 513.31

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.

BODIPY™ FL Sulfonated Succinimidyl Ester (Invitrogen™)

The amine-reactive BODIPY® FL sulfosuccinimidyl ester can be used to create green-fluorescent bioconjugates. This reactive moiety is sulfonated to increase water solubility. The electronically neutral BODIPY® FL dye has the most fluorescein-like spectra of the green-fluorescent BODIPY® dyes.

Pacific Orange™ 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 Orange™ has an excitation and emission maximum of 400/551 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.

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.

pHrodo™ Red Microscale Labeling Kit (Invitrogen™)

pHrodo® Red is the new name for pHrodo®, a fluorogenic dye that dramatically increases in fluorescence as the pH of its surroundings become more acidic. Original pHrodo® and pHrodo® Red dyes are the same molecule. Use this pHrodo® Red microscale protein labeling kits to conveniently label 3 separate samples of small amounts (20-100 µg) of purified protein or antibody with pHrodo®-SE conjugate.

pHrodo® Red dye conjugates are non-fluorescent outside the cell, but fluoresce bright red in phagosomes, making them ideal tools for studies ranging from phagocytosis of bioparticles to receptor internalization. pHrodo Red™ offers staining that is faster and more accurate than ratiometric dyes.

The pHrodo® Red Microscale Labeling Kit Is:
Convenient—Labeling typically takes 2 hours with less than 30 min hands-on
Versatile—Label proteins with MW between 12 and 150 kDa
Customizable—Create your own probes for studying phagocytosis and endocytosis

Get Better Results in Endocytosis Studies
Endocytosis and phagocytosis underly physiological events ranging from host-pathogen response to receptor desensitization via internalization (as is common in the GPCR class of receptors). Once internalized, material can be acidified in endosomes or lysosomes. When conjugated to target molecules, our pHrodo® Red dye fluorogenic pH sensitive probe allows direct visual monitoring of material entering these acidic compartments. At neutral and basic pH values, pHrodo® Red dye emission is at its minimum; with acidification, pHrodo® Red dye emission is increased by as much as 8-fold (at pH 4). The fluorescence of pHrodo® Red dye is dimmed by complete media and whole blood, further improving the signal-to-noise ratio. The pHrodo® Red Microscale Labeling Kit can label proteins with molecular weights between 12 and 150 kDa, including IgG antibodies (~150 kDa) in quantities ranging from 20-100 µg.

Gain Experimental Flexibility with a Validated Technology
pHrodo® has been cited in over 150 publications and used in a variety of applications. It is validated in flow cytometry, high throughput and high content screening (HTS and HCS), and other imaging applications. pHrodo® Red offers reduced signal variability and improved timing compared to ratiometric dyes like BCECF and SNARF, and the lack of fluorescence outside the cell eliminates the need for wash steps and quencher dyes. The red fluorescence of pHrodo® Red is ideal for multiplexing applications with green-fluorescent compounds such GFP, Fluo-4, or calcein.

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

Oregon Green™ 488 Cadaverine, 5-isomer (Invitrogen™)

The primary aliphatic amine of the green fluorescent Oregon Green® cadaverine 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 biotinylated 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). This molecule can also be used as a water-soluble, fixable polar tracer.

SiteClick™ Alexa Fluor™ 488 sDIBO Alkyne (Invitrogen™)

SiteClick labeled sDIBO alkynes for antibody labeling are optimized for easy attachment to azido modified antibodies using copper-free Click chemistry. This labeled sDIBO alkyne can be used with antibodies that have been modified using the SiteClick Antibody Azido Modification Kit or engineered to contain azido moieties. These labeled sDIBO alkynes are improved versions of our original DIBO cyclooctynes, yielding conjugates that are less "sticky" and that produce lower signal background in biological samples. This modular labeling system gives you the option to choose different fluorescent labels for your antibody and attach another molecule via streptavidin or your own molecule via amine-reactive or amine-containing moieties depending on your assay.

Learn more about SiteClick labeling technology ›

Custom SiteClick Antibody Labeling Service and sDIBO labels
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, chelates for PET imaging, and many others.

Alexa Fluor™ 350 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.

SiteClick™ Alexa Fluor™ 647 sDIBO Alkyne (Invitrogen™)

SiteClick labeled sDIBO alkynes for antibody labeling are optimized for easy attachment to azido modified antibodies using copper-free Click chemistry. This labeled sDIBO alkyne can be used with antibodies that have been modified using the SiteClick Antibody Azido Modification Kit or engineered to contain azido moieties. These labeled sDIBO alkynes are improved versions of our original DIBO cyclooctynes, yielding conjugates that are less "sticky" and that produce lower signal background in biological samples. This modular labeling system gives you the option to choose different fluorescent labels for your antibody and attach another molecule via streptavidin or your own molecule via amine-reactive or amine-containing moieties depending on your assay.

Learn more about SiteClick labeling technology ›

Custom SiteClick Antibody Labeling Service and sDIBO labels
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, chelates for PET imaging, and many others.

SiteClick™ Biotin sDIBO Alkyne (Invitrogen™)

SiteClick labeled sDIBO alkynes for antibody labeling are optimized for easy attachment to azido modified antibodies using copper-free Click chemistry. This labeled sDIBO alkyne can be used with antibodies that have been modified using the SiteClick Antibody Azido Modification Kit or engineered to contain azido moieties. These labeled sDIBO alkynes are improved versions of our original DIBO cyclooctynes, yielding conjugates that are less "sticky" and that produce lower signal background in biological samples. This modular labeling system gives you the option to choose different fluorescent labels for your antibody and attach another molecule via streptavidin or your own molecule via amine-reactive or amine-containing moieties depending on your assay.

Learn more about SiteClick labeling technology ›

Custom SiteClick Antibody Labeling Service and sDIBO labels
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, chelates for PET imaging, and many others.

Pierce™ Streptavidin, Hydrazide-Activated (Thermo Scientific™)

Thermo Scientific Pierce Hydrazide-Activated Streptavidin conjugate include recombinant streptavidin in a purified form activated for crosslinking to carbonyl groups in a molecule.

Related Products
Pierce™ Streptavidin
Pierce™ Streptavidin, Horseradish Peroxidase Conjugated
Pierce™ Streptavidin, Alkaline Phosphatase Conjugated
Pierce™ Streptavidin, Maleimide-Activated

DyLight™ 755 Microscale Antibody Labeling Kit (Thermo Scientific™)

The Thermo Scientific DyLight 755 Microscale 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 microscale kit contains all of the necessary components to perform five separate labeling reactions using 100 µg of IgG—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 Antibody Labeling Kit

Zenon™ Alexa Fluor™ 555 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

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.

Biotin-XX Microscale Protein Labeling Kit with FluoReporter™ Biotin Quantitation Assay Kit (Invitrogen™)

Microscale Protein Labeling Kits provide a convenient means for attaching a fluorescent label to a small amount of antibody or protein (20–100 μg). The kits are available in four Alexa Fluor® colors (or biotin) and supply everything needed for three labeling and separation reactions.

Important Features of Microscale Protein Labeling Kits:

• Labeled proteins typically ready to use typically in 2 hr (~30 min hands-on time)
• Optimized for 20–100 μg of protein with molecular weights between 12 and 150 kDa
• Purified using convenient spin filters with yields between 60 and 90%
• Stabilizing proteins must be removed from the sample before labeling


Stable Reaction Chemistry and Superior Alexa Fluor® Dyes
In the Microscale Protein Labeling Kits, the reactive dye contains a tetrafluorophenyl (TFP) ester moiety that is more stable in solution than the commonly used succinimidyl (NHS) ester. TFP esters react efficiently with primary amines of proteins to form stable dye–protein conjugates. 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.

Click-IT™ L-Homopropargylglycine (HPG) (Invitrogen™)

Click-iT® homopropargylglycine (HPG) provides a fast, sensitive, non-toxic and most importantly non-radioactive alternative to the traditional technique,35S methionine for the detection of nascent protein synthesis. HPG is an amino acid analog that contains a very small modification, specifically an alkyne moiety that can be fed to cultured cells and incorporated into proteins during active protein synthesis. Detection utilizes the chemoselective ligation or “click" reaction between and azide and an alkyne where the alkyne modified protein is detected with an azide-containing dye or hapten together with either the Click-iT® Cell Reaction Buffer Kit or the Click-iT® Protein Buffer Kit. With the Click-iT® Cell Reaction Buffer Kit, cells can be analyzed by fluorescence microscopy, flow cytometry or high-content imaging and analysis. With the Click-iT® Protein Reaction Buffer Kit, achieve detection sensitivity in 1-D gels and western blots in the low femtomole range or perform LC-MS⁄MS and MALDI MS analysis.

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.

Qdot™ 585 ITK™ Carboxyl Quantum Dots (Invitrogen™)

Qdot® 585 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® 585 ITK™ carboxyl quantum dot has emission maxima of ~585 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.

Alexa Fluor™ 532 NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor® 532 is a bright yellow 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 NHS ester (or succinimidyl ester) of Alexa Fluor® 532 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® 532 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 530/555 nm
Extinction coefficient: 81,000 cm-1M-1
Molecular weight: 723.8

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.

Alexa Fluor™ 532 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.

View a selection guide for all Protein Labeling Kits.

View the Fluorophore Selection Guide.

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.

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.

Oregon Green™ 488 Iodoacetamide, mixed isomers (Invitrogen™)

The thiol reactive Oregon Green® 488 iodoacetamide can be used to can be used to create green fluorescent bioconjugates with excitation/emission maxima ~496/524 nm.
This fluorinated analog of fluorescein overcomes some of the key limitations of fluorescein, including greater photostability and a lower pKa (pKa ~ 4.7 versus 6.4 for fluorescein), making its fluorescence essentially pH insensitive in the physiological pH range.

DyLight™ 680 Antibody Labeling Kit (Thermo Scientific™)

The Thermo Scientific DyLight 680 Antibody Labeling Kit contains 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. 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 680 NHS-ester in convenient single-use vials, as well as purification resin and spin columns for the preparation of ready-to-use conjugate..

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 a stand-alone reagent.

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)
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 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.

Related Products
DyLight™ 680 NHS-Ester
DyLight™ 680 Microscale Antibody Labeling Kit

Zenon™ R-Phycoerythrin Mouse IgG2a 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™ Alexa Fluor™ 647 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

Click-iT™ Amine sDIBO Alkyne for Antibody Labeling (Invitrogen™)

The Click-iT Amine sDIBO Alkyne for Antibody Labeling is optimized for easy attachment to azido modified antibodies using copper-free Click chemistry. This sDIBO label can be used with antibodies that have been modified using the SiteClick Antibody Azido Modification Kit or antibodies that have been engineered to contain azido moieties. These sDIBO alkynes are improved versions of our original DIBO cyclooctynes, yielding conjugates that are less "sticky" and give lower signal background in biological samples.

This modular labeling system gives you the option to choose different fluorescent labels for your antibody and attach another molecule via streptavadin or your own molecule via amine-reactive or amine-containing moieties depending on your assay.

There are multiple Click-iT sDIBO labels to choose from:
Click-iT Alexa Fluor 488 sDIBO Alkyne for Antibody Labeling
Click-iT Alexa Fluor 555 sDIBO Alkyne for Antibody Labeling
Click-iT Alexa Fluor 647 sDIBO Alkyne for Antibody Labeling
Click-iT Biotin sDIBO Alkyne for Antibody Labeling
Click-iT Amine sDIBO Alkyne for Antibody Labeling
Click-iT SDP Ester sDIBO Alkyne for Antibody Labeling

Learn more about SiteClick labeling technology ›

Custom SiteClick Antibody Labeling Service and sDIBO labels
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, chelates for PET imaging, and many others.

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.

2-(2,3-Naphthalimino)ethyl Trifluoromethanesulfonate (Invitrogen™)

2-(2,3-Naphthalimino)ethyl trifluoromethanesulfonate reacts rapidly with the anions of carboxylic acids in acetonitrile to give adducts that are reported to be detectable by absorption at 259 nm down to 10 femtomoles and by fluorescence at 394 nm down to femtomoles.

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.

Zenon™ pHrodo™ iFL Green Mouse IgG Labeling Reagent (Invitrogen™)

Zenon labeling technology provides a fast, versatile, and reliable method for adding a fluorescent label to an antibody. Only a small amount of starting material is needed, and the method is optimized for efficient labeling of antibodies in serum, ascites fluid, or hybridoma suspensions.

Combined with pHrodo iFL dye, this technology provides a rapid, scalable, and easy way to determine how well an antibody internalizes into target cells. pHrodo iFL Green dye dramatically increases its fluorescence as the pH of its surroundings becomes more acidic.

• Labeled antibodies typically ready to use in 10 minutes
• Simple, no purification required
• Fast, accurate internalization results—no need for wash steps or quenchers

Preserve primary antibody function and affinities
Reactive dye labeling of primary antibodies can have unpredictable and undesirable outcomes. One of these is reduced binding affinity due to label addition in the binding pocket. The Zenon antibody labeling approach, which is targeted to the Fc tail, avoids this outcome.

Custom antibody conjugates
If the antibody conjugate you need is not available as one of our stocked products, we will 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.

Alexa Fluor™ 633 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.

EZ-Link™ Activated Peroxidase Antibody Labeling Kit (Thermo Scientific™)

The Thermo Scientific Pierce Activated Peroxidase and Antibody Labeling Kit is designed to prepare and purify HRP-conjugated antibodies with any previously unmodified primary or secondary antibody (IgG).

Features of the Activated Peroxidase and Antibody Labeling Kit:

Activated HRP – glutaraldehyde-activated horseradish peroxidase, ready for conjugation to antibodies and other proteins at sites of primary amines (e.g., lysines)
Permanent conjugation – reacts with primary amines to form covalent amide bonds; no reduction step is necessary to secure the linkage
High activity HRP – enzyme activity is greater than 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 in the kit is sufficient for reaction with 0.3 mg of IgG to produce about 0.5 mL of conjugate

Activated Peroxidase is HRP enzyme that has been activated with glutaraldehyde to contain reactive groups that will spontaneously conjugate with amines on an antibody (or other protein). Incubation of this Activated Peroxidase with IgG in sodium carbonate buffer (pH 9.5) results in formation of a conjugate where both the enzyme activity of HRP and the antigen-binding activity of IgG are preserved. The Antibody Labeling Kit includes the Activated Peroxidase and a Protein A/G column and buffers to purify the antibody conjugate from excess, unreacted HRP enzyme.

For additional information about glutaraldehyde conjugation, see our review of reductive amination and the discussion of applications that follows.

5-SFX (6-(Fluorescein-5-Carboxamido) Hexanoic Acid, Succinimidyl Ester), single isomer (Invitrogen™)

Searching for superior alternatives to fluorescein? Our Alexa Fluor Dye Series offers everything you're looking for and more.

DyLight™ 680 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 680 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 680 used to fluorescently label sulfhydryl-containing peptides, proteins and other biomolecular probes.

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.

Features of DyLight 680 Maleimide:

High performance—DyLight 680 fluoresces brighter than Alexa Fluor 680 and Cy5.5 dyes
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 680 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.

EZ-Link™ Micro Sulfo-NHS-Biotinylation Kit (Thermo Scientific™)

The Thermo Scientific EZ-Link Micro Sulfo-NHS-Biotinylation Kit contains reagents sufficient for 8 biotinylation reactions (e.g., 0.05–0.2 mg antibody per reaction). The EZ-Link Sulfo-NHS-Biotin reagent included in the kit is a short-chain, water-soluble biotinylation reagent used for labeling antibodies, proteins and other molecules that have primary amines.

Features of EZ-Link Sulfo-NHS-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 lysine side-chains 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
Very short—spacer arm (total length added to target) is 13.5 angstroms; it consists of the native biotin valeric acid group only.

Sulfo-NHS-Biotin is the shortest 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. Sulfo-NHS-Biotin is offered in several package sizes and as complete protein labeling kits.

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.

Related Products
EZ-Link™ Sulfo-NHS-Biotin
EZ-Link™ Sulfo-NHS-Biotinylation Kit

Alkyne, Succinimidyl Ester (3-Propargyloxypropanoic Acid, Succinimidyl Ester) (Invitrogen™)

Conjugates prepared with the amine-reactive alkyne, succinimidyl ester can be detected with an azide-containing molecule in a click chemistry 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.

Alexa Fluor™ 647 Microscale Protein Labeling Kit (Invitrogen™)

Microscale Protein Labeling Kits provide a convenient means for attaching a fluorescent label to a small amount of antibody or protein (20–100 µg). The kits are available in four Alexa Fluor® colors (or biotin) and supply everything needed for three labeling and separation reactions.

Important Features of Microscale Protein Labeling Kits:
• Labeled proteins typically ready to use typically in 2 hours (~30 minutes hands-on time)
• Optimized for 20–100 µg of protein with molecular weights between 12 and 150 kDa
• Purified using convenient spin filters with yields between 60 and 90%
• Stabilizing proteins must be removed from the sample before labeling

Stable Reaction Chemistry and Superior Alexa Fluor® Dyes
In the Microscale Protein Labeling Kits, the reactive dye contains a succinimidyl (NHS) ester moiety that reacts with primary amines of proteins to form stable dye-protein conjugates. 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.

PowerLoad™ Concentrate, 100X (Invitrogen™)

PowerLoad™ is an optimized formulation of nonionic, Pluronic® surfactant polyols for the solubilization of water-insoluble dyes and other materials in physiological media. These surfactants, for instance Pluronic® F-127, have been used to help disperse acetoxymethyl (AM) esters of fluorescent ion indicators such as fluo-4, fura-2, indo-1, fluo-3, and SBFI; they appear to be required for loading of other dyes (e.g. SBFI-AM or PBFI-AM). The use of PowerLoad™ is optional with red shifted calcium indicators and other large molecular weight AM ester dyes, and may also be useful for dispersing other lipophilic probes. The concentration of Pluronic® surfactants in PowerLoad™ is less than 0.2%. PowerLoad™ is effective in combination with water soluble Probenecid (P36400) to aid AM ester dye-loading and retention in cells that actively extrude the de-acetylated form through anion pumps. Together, these reagents allow for maximal loading of dyes with a minimum of effort in both imaging and high throughput screening (HTS) applications. Appropriate controls should be performed to make certain that PowerLoad™ is not altering the membrane properties of the cell.

5-IAF (5-Iodoacetamidofluorescein) (Invitrogen™)

The thiol-reactive 5-iodoacetamidofluorescein (5-IAF) can be used to produce bioconjugates with the 5-isomer of fluorescein.

Alexa Fluor™ 488 Hydroxylamine (Invitrogen™)

Alexa Fluor® 488 Hydroxlamine is useful as a cell tracer and as a reactive dye for labeling aldehydes or ketones in polysaccharides or glycoproteins. Alexa Fluor® 488 is a bright, green fluorescent dye with excitation ideally suited to the 488 nn 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® hydroxlamine:

• Fluorophore label : Alexa Fluor® 488 dye
• Reactive group: hydroxlamine
• Reactivity: Aldehydes or ketones
• Ex/Em of the conjugate: 494/518 nm
• Extinction coefficient: 77,000 cm-1M-1
• Spectrally similar dyes: FITC, GFP
• Molecular weight: 895.07

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)

7-Diethylaminocoumarin-3-Carboxylic Acid, Succinimidyl Ester (Invitrogen™)

The amine-reactive coumarin, 7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester can be used to create blue-fluorescent bioconjugates. When compared with AMCA conjugates, conjugates of the UV-light-excitable 7-dialkylaminocoumarin fluorophore have slightly longer-wavelength emission spectra (~470 nm).

Qdot™ 705 ITK™ Amino (PEG) Quantum Dots (Invitrogen™)

Qdot® 705 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® 705 ITK™ amino quantum dot has emission maxima of ~705 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.

Fluorescein-5-Thiosemicarbazide (Invitrogen™)

The amine-containing fluorescein-5-thiosemicarbazide can be reversibly coupled to aldehydes and ketones to form a Schiff base - which can be reduced to a generate stable amine derivative by sodium borohydride (NaBH4) or sodium cyanoborohydride (NaCNH3). 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).

EZ-Link™ Maleimide-PEG11-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link Maleimide-PEG11-Biotin is a long, maleimide-activated, sulfhydryl-reactive biotinylation reagent that includes an 11-unit polyethylene glycol spacer arm for increased water-solubility and reach.

Features of EZ-Link Maleimide-PEG11-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, 11-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
Long—spacer arm (total length added to target) is 59.1 angstroms

Maleimide-PEG11-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, 11-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.

Qdot™ 525 ITK™ Carboxyl Quantum Dots (Invitrogen™)

Qdot® 525 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® 525 ITK™ carboxyl quantum dot has emission maxima of ~525 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.

CPM (7-Diethylamino-3-(4'-Maleimidylphenyl)-4-Methylcoumarin) (Invitrogen™)

The thiol-reactive coumarin, CPM is very weakly fluorescent until reacted with thiols producing a conjugate with excitation/emission maxima of ~384/470 nm.

SiteClick™ Qdot™ 655 Antibody Labeling Kit (Invitrogen™)

Create a perfectly labeled antibody with the SiteClick™ Qdot® 655 Antibody Labeling Kit. This kit replaces the conventional Qdot® 655 Antibody Conjugation Kit (Q22021MP). 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® 655 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® 655 labels can be used in confocal or traditional fluorescence microscopy.
• In flow cytometry, Qdot® 655 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.

DyLight™ 488 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 488 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 488 used to fluorescently label sulfhydryl-containing peptides, proteins, and other biomolecular probes.

DyLight 488 has high fluorescence intensity over a broad pH range (pH 4-9) and is more photostable than Cy2™ dye, 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.

Features of DyLight 488 Maleimide:

High performance—DyLight 488 shows brighter fluorescence than Alexa Fluor 488, FITC and Cy2 dye
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 488 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.

Alexa Fluor™ 532 Antibody Labeling Kit (Invitrogen™)

Molecular Probes® Alexa Fluor® Antibody Labeling Kits provide a convenient means to label small amounts of antibodies with Alexa Fluor® dyes (choice of 10 colors). This kit is optimized for labeling 100 µg of antibody per reaction with green-yellow fluorescent Alexa Fluor® 532. Comparably small amounts of other proteins (>40 kDa) can also be labeled.

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 Alexa Fluor® 532 Antibody Labeling Kit:
• With an excitation and emission maximum of 530/554 nm, Alexa Fluor® 532 can be efficiently excited using a 532 nm Nd:YAG laser line and detected under standard Rhodamine 6G filters
• Labeled proteins typically ready to use typically 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.

Superior 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 for use in diagnostic procedures.

Alexa Fluor™ 488 Hydrazide (Invitrogen™)

Alexa Fluor® 488 Hydrazide is useful as a cell tracer and as a reactive dye for labeling aldehydes or ketones in polysaccharides or glycoproteins. Alexa Fluor® 488 is a bright, green fluorescent dye with excitation ideally suited to the 488 nn 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® hydrazide:

• Fluorophore label : Alexa Fluor® 488 dye
• Reactive group: hydrazide
• Reactivity: Aldehydes or keytones in polysaccharides or glycoproteins
• Ex/Em of the conjugate: 493/517 nm
• Extinction coefficient: 71,000 cm-1M-1
• Spectrally similar dyes: FITC, GFP
• Molecular weight: 570.48

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)