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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.

Alexa Fluor™ 594 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 red fluorescent Alexa Fluor® 594. 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® 594 Antibody Labeling Kit:
• With an excitation and emission maximum of 590/617 nm, Alexa Fluor® 594 can be efficiently excited using a 594 nm Kr or He-Ne laser line and detected under standard Texas Red® 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 Zor 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.

DSB-X™ Biotin 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.

DyLight™ 488 Antibody Labeling Kit (Thermo Scientific™)

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

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

Features of the DyLight 488 NHS Ester:

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

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

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

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

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

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

FluoReporter™ Cell-Surface Biotinylation Kit (Invitrogen™)

The FluoReporter® Cell-Surface Biotinylation Kit provides a convenient method to label proteins exposed on the cell surface including, but not limited to, membrane proteins. The kit supplies 5 vials of biotin-XX SSE, and each vial can be used for several 1-mL labeling reactions (containing 2.5 x 107 cells/mL).

Important Features of FluoReporter® Protein Labeling Kits:

• Biotinylation is typically complete in under 1 hour
• Optimized for 1 mL labeling reactions each containing 2.5 x 107 cells/mL
• Biotinylated proteins can be subsequently identified using western blot techniques and labeled avidin and streptavidin conjugates (find an anti-biotin antibody using our Secondary Antibody Selector Tool)


Learn More About Protein Labeling
We offer a wide selection of Molecular Probes® protein and antibody 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.

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

Zenon™ Allophycocyanin 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.

DyLight™ 350 Antibody Labeling Kit (Thermo Scientific™)

The Thermo Scientific DyLight 350 Antibody Labeling Kit contains an NHS ester-activated derivative of high-performance DyLight 350 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.

DyLight 350 is a derivative of aminomethylcoumarine acetate (AMCA) and has a higher fluorescence intensity than Alexa Fluor™ 350 and AMCA in many applications. The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates. DyLight 350 Amine-Reactive Dye is also available as a stand-alone reagent.

Features of DyLight 350 NHS Ester:

High performance—DyLight 350 shows brighter fluorescence than Alexa Fluor 350 and AMCA
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 350 Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

Typical labeling reactions require DyLight 350 Amine-Reactive Dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to specific amounts of the labeling reagent. This feature allows DyLight 350 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

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DyLight™ 350 NHS Ester
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SiteClick™ Antibody Azido Modification Kit (Invitrogen™)

Create a label-ready site-specific azido-modified antibody without lengthy and inefficient genetic modification using the SiteClick Antibody Azido Modification Kit. SiteClick labeling uses enzymes to specifically attach an azido moiety to the heavy chains of an IgG antibody, ensuring that the antigen binding domains remain unaltered for binding to the antigen target. This site selectivity is achieved by targeting the carbohydrate domains present on essentially all IgG antibodies regardless of isotype and host species. Once azido–modified, a variety of sDIBO alkyne labels are available for attachment to the antibody via Click chemistry (see list below). This provides the flexibility to choose different labels for your antibody depending on your assay.

Features of the SiteClickAntibody Azido Modification Kit:
• Contains everything required to label 100–250 µg of IgG antibody
• Easy-to-follow step-by-step protocol
• Highly efficient, site-specific, reproducible labeling chemistry results in high quality antibody conjugate

Learn more about SiteClick labeling technology ›

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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

Custom SiteClick Antibody Labeling Service
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.

Click-IT™ Tetramethylrhodamine (TAMRA) Protein Analysis Detection Kit (Invitrogen™)

The Click-iT® Tetramethylrhodamine (TAMRA) Glycoprotein Detection Kit provides the second part of the simple and robust two-step technique to identify and characterize glycoproteins by one- or two-dimensional gel electrophoresis. In step two, after the incorporation of the azide handle into protein glycan structures with either a Click-iT® metabolic labeling reagent or the Click-iT® Enzymatic Labeling system, the azide-modified glycoproteins are detected via the chemoselective ligation or click reaction between an azide and an alkyne. Gels with TAMRA labeled glycoproteins can be subsequently stained with the Multiplexed Proteomics™ technologies, SYPRO® Ruby total protein stain and PRO-Q® Emerald glycoprotein stain for the differential analysis of glycoprotein subclasses, total proteins and total glycoproteins in the same gel. Click-iT® modified glycoproteins are also compatible with downstream LC-MS/MS and MALDI MS analysis for identification.

EZ-Link™ Micro NHS-PEG4-Biotinylation Kit (Thermo Scientific™)

The Thermo Scientific EZ-Link NHS-PEG4 Biotinylation Kit contains EZ-Link NHS-PEG4-Biotin and accessory reagents required for 8 0.05–0.2 mg protein labeling reactions. EZ-Link NHS-PEG4-Biotin is a pegylated, water-soluble reagent for simple and efficient biotin labeling of antibodies, proteins and other primary amine-containing macromolecules.

Features of EZ-Link NHS-PEG4-Biotin:

Protein labeling—biotinylate antibodies or other proteins for detection or purification using streptavidin probes or resins
Amine-reactive—reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the amino-termini of polypeptides
Pegylated – spacer arm contains a hydrophilic, 4-unit, polyethylene glycol (PEG) group
Enhances solubility – pegylation imparts water solubility to the biotinylated molecule, helping to prevent aggregation of biotinylated antibodies stored in solution
Irreversible—forms permanent amide bonds; spacer arm cannot be cleaved
Long reach – spacer arm (total length added to target) is 29 angstroms; this reduces steric hindrance when binding to avidin molecules

NHS-PEG4-Biotin is a long (29.0Å), pegylated, water-soluble, NHS-ester biotinylation reagent to label amines and maximize solubility of antibodies and other proteins. The N-hydroxysuccinimide ester (NHS) group reacts specifically and efficiently with lysine and N-terminal amino groups to form stable amide bonds. The hydrophilic polyethylene glycol (PEG) spacer arm imparts water solubility that is transferred to the biotinylated molecule, thus reducing aggregation of labeled proteins stored in solution. The PEG spacer arm also gives the reagent a long and flexible connection to minimize steric hindrance for binding to avidin molecules.

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

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

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

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CellTrace™ Calcein Red-Orange, AM - Special Packaging (Invitrogen™)

CellTrace calcein red-orange AM is a cell-permeant dye that can be used to determine cell viability in most eukaryotic cells. Unlike calcein AM (C-1430, C-3099, C-3100), CellTrace calcein red-orange AM is intrinsically fluorescent; thus, an additional wash step may be necessary to minimize background fluorescence from dye that is not taken up by cells. However, CellTrace calcein red-orange (excitation/emission maxima 577/590 nm) is well-retained by live cells that possess intact plasma membranes, and consequently it is a useful cell tracer and indicator of cell viability.

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

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

The NHS ester (or succinimidyl ester) of BODIPY® FL is the most popular tool for conjugating the dye to a protein or antibody. NHS esters can be used to label the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting BODIPY® FL 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® FL NHS ester:

Fluorophore label: BODIPY® FL dye
Reactive group: NHS ester (succinimidyl ester)
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 502/510 nm
Extinction coefficient: 82,000 cm-1M-1
Molecular weight: 389.16

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.

5-FAM (5-Carboxyfluorescein), single isomer (Invitrogen™)

The single isomer, 5-FAM, contains a carboxylic acid that can be used to react with primary amines via carbodiimide activation of the carboxylic acid. Fluorescein is the most common fluorescent derivatization reagent for labeling biomolecules. In addition to its relatively high absorptivity, excellent fluorescence quantum yield, and good water solubility, fluorescein has an excitation maximum that closely matches the 488 nm spectral line of the argon-ion laser.

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

Thermo Scientific™ Pierce™ Premium Grade Sulfo-NHS-SS-Biotin is our highest quality formulation of this amine-reactive, cleavable biotinylation reagent, specially characterized for applications where product integrity and risk minimization are paramount. Sulfo-NHS-SS-biotin is used for cleavable attachment of biotin to protein and cell surface amines.

• High quality—identity and purity confirmed by multiple 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 help 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-SS-Biotin provides more clearly defined quality and product support by including increased analytical testing and product characterization, greater batch-specific information and quality assurance review, extensive lot sample retention, and change control notification.

Sulfo-NHS-SS-biotin is a water-soluble, NHS-ester biotinylation reagent with a spacer arm that includes a cleavable disulfide bond for reversible labeling of proteins and cell surface primary amines. This reagent is water-soluble, enabling biotinylation in the absence of organic solvents such as DMSO or DMF. The compound is particularly useful for labeling and purifying cell surface proteins because its sulfonate group prevents it from permeating cell membranes and its cleavable spacer arm enables initially biotinylated proteins to be released from streptavidin affinity columns.

Zenon™ Biotin-XX Mouse IgG2a 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

R-Phycoerythrin (Thermo Scientific™)

Thermo Scientific R-Phycoerythrin is a red-fluorescent, multi-subunit protein that can be crosslinked or conjugated to other molecules to make fluorescent probes.

Features of R-Phycoerythrin:

R-Phycoerythrin—red-fluorescent, multi-subunit protein in the phycobiliprotein family
Large Stokes shift —excitation (absorption) at one of three wavelengths produces light emission at a much higher wavelength

This fluorochrome is a member of the phycobiliprotein family of proteins isolated from the marine algae Porphyra tenera or Gastroclonium coulterii. Fluorochrome-labeled reagents have high resolution and are therefore advantageous in immunological assays, such as live cell staining, double-labeling techniques and cell sorting procedures. The strong absorption bands of the R-Phycoerythrin are in the visible region of the spectrum, extending from the green to the far-red wavelengths. The absorbance spectra extends over a broad range of potential excitation wavelengths, allowing for versatility in the excitation source and creating large Stokes shifts, thus minimizing interference from Rayleigh-scattered light.

Properties of R-Phycoerythrin:

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Zenon™ Horseradish Peroxidase Rabbit IgG Labeling Kit (Invitrogen™)

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

Important Features of Zenon® Labeling Technology:

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


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

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

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

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

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

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

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

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

Related Links:

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

Click-iT™ HPG Alexa Fluor™ 594 Protein Synthesis Assay Kit (Invitrogen™)

The Click-iT® HPG Alexa Fluor® 594 Protein Synthesis Assay Kit provides a fast, sensitive, non-toxic, and non-radioactive method for the detection of nascent protein synthesis utilizing fluorescence microscopy, high-content imaging, or flow cytometry. Included in the kit are L-homopropargylglycine (HPG), an amino acid analog of methionine containing an alkyne moiety, and Alexa Fluor® 594 azide. The HPG is fed to cultured cells and incorporated into proteins during active protein synthesis. Addition of the Alexa Fluor® 594 azide leads to a chemoselective ligation or “click" reaction between the green fluorescent azide and the alkyne, allowing the modified proteins to be detected by imaged-based analysis.

Non-radioactive alternative—an alternative to the traditional 35S-methionine
Visualize bulk protein dynamics—fluorescent tagging of proteins allows their localization to be determined, including aggregation
Specificity—selective, specific reaction between label and detection tags
Stability —product contains an irreversible, covalent bond
Multiplex-enabled—use in conjuction with Click®-iT AHA (azide amino acid and alkyne dye) to detect spatial and temporal differences
Applicability to biological samples—easy detection; high sensitivity and low background, regardless of complexity

The Click-iT® HPG Alexa Fluor® 594 Protein Synthesis Assay Kit has been successfully tested in HeLa, A549, and U-2 OS cells with a variety of reagents that inhibit protein synthesis, including cycloheximide and anisomycin. The applicability of these probes to monitor protein degradation has also been shown using inhibitors of the proteasome (MG132 and Bortezomib) and blockers of autophagy (chloroquine) in HeLa cells.

Additionally, due to differences in Click-iT® chemistry between the Click-iT® HPG Alexa Fluor® 594 Protein Synthesis Assay Kit and Click-iT® AHA Alexa Fluor® 488 HCS Kit, these kits can be used in conjunction for spatial or temporal determination of differences in nascent protein synthesis.

Click-IT™ Myristic Acid, Azide (12-Azidododecanoic Acid) (Invitrogen™)

Identify and characterize myristylated proteins with Click-iT® myristic acid, azide using the powerful click chemistry, a simple and robust two-step labeling and detection technique. In step one, the azide-containing biomolecule is fed to cells or animals and actively incorporated into proteins. Unlike other labels such as biotin or a fluorescent dye, the azide-tag is small enough that the tagged molecule is an acceptable substrate for the enzymes that incorporate this building block into proteins. Detection utilizes the chemoselective ligation or “click" reaction between and azide and an alkyne where the modified protein is detected with the corresponding alkyne-containing dye or hapten using 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 (HCS) together with other biomarkers of interest for content and context rich results. 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.

2',7'-Difluorofluorescein (Oregon Green™ 488) (Invitrogen™)

Oregon Green® 488 can be used as a reference standard for Oregon Green® 488 conjugates or a pH sensor of moderately acidic solutions (pKa ~4.7)

Zenon™ Alexa Fluor™ 647 Goat IgG Labeling Kit - 50 Labeling (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

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

Thermo Scientific EZ-Link Iodoacetyl-LC-Biotin is a mid-length, haloacetyl-activated, sulfhydryl-reactive biotinylation reagent that forms stable, irreversible thioether bonds at alkaline pH.

Features of EZ-Link Iodoacetyl-LC-Biotin:

Protein labeling—biotinylate antibodies or other proteins for use in protein methods
Membrane-permeable—can be used to label inside cells (intracellular)
Thiol-reactive—reacts with sulfhydryls (-SH), such as the side-chain of cysteine (C)
Iodoacetyl-activated—perform reactions in the dark at pH 7.5 to 8.5 in Tris or borate buffer
Irreversible—forms permanent thioether bonds; spacer arm cannot be cleaved
Solubility—must be dissolved in DMSO or DMF before further dilution in aqueous buffers
Medium length—spacer arm (total length added to target) is 27.1 angstroms; contains hexylenediamine extension

Iodoacetyl-LC-Biotin is a haloacetyl-biotin compound for labeling protein cysteines and other molecules that contain sulfhydryl groups. This reagent specifically reacts with reduced thiols (-SH) in alkaline buffers to form permanent (irreversible) thioether bonds. The unique feature of Iodoacetyl-LC-Biotin is its extended yet chemically simple hexylenediamine spacer arm.

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. Iodoacetyl reagents specifically react with sulfhydryl groups (-SH) at pH 8.3 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.

Applications:
• Electron microscopy studies on spatial relationships between proteins (Ref.1)
• Localizing the SH1 thiol of the myosin head using avidin-biotin complexes in electron microscopy (Ref.2)

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

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

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

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.

GalNAz (N-azidoacetylgalactosamine tetraacylated) (Thermo Scientific™)

Thermo Scientific Pierce GalNAz (N-azidoacetylgalactosamine-tetraacylated) is an azide-labeled sugar that provides a highly specific approach for studying glycoproteins through in vivo metabolic labeling and chemoselective ligation.

Features of Azido-Sugars:

Bioorthogonal—the azido group is small, nonreactive and absent from living systems; as such the azido-sugar compounds do not interfere with endogenous cellular pathways and substitute for their naturally occurring analogs
Compatible—reaction chemistry with phosphine compounds occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents
Chemoselective—azide and phosphine groups do not react or interfere with components of biological samples but conjugate to one another with high efficiency
Versatile—azide tag can be targeted for detection, immobilization, conjugation or affinity purification depending on which phosphine-activated compound it is reacted with

These sugars are azide-derivatives of naturally occurring monosaccharides that cells use to glycosylate proteins using post-translational modification biochemical pathways. The azide functional group is small and nonreactive with endogenous molecules. When supplied to cells, these compounds become incorporated by glycosylation events to effectively "tag" glycoproteins with the azide group. The azide group then can be specifically targeted for detection or conjugation using alkyne-activated reagents ("click" chemistry) or phosphine-activated reagents (Staudinger ligation).

When used in combination with phosphine-activated fluorescent dyes, biotin reagents, and or other compounds, these azido-modified sugars facilitate the investigation of cellular pathways involving glycosylation.

There are several classes of glycoproteins grouped by the type of carbohydrate and amino acid linkage site. N-linked glycosylation is a modification of asparagine amines, whereas O-linked glycosylation occurs through the hydroxyl of serine and threonine residues. The azido-modified sugars are metabolic substitutes for endogenous amino sugars. ManNAz is converted by cells to an azido sialic acid derivative that is used for N-linked glycosylation of cell surface proteins. GlcNAz and GalNAz are predominantly used to label the O-linked glycosylation (O-GlcNAc and O-GalNAc).

Related Products
GlcNAz (N-azidoacetylglucosamine tetraacylated)
ManNAz (N-azidoacetylmannosamine tetraacylated)

Click-iT™ Protein Enrichment Kit, for click chemistry capture of azide-modified proteins (Invitrogen™)

The Click-iT® Protein Enrichment Kit for the efficient capture of click chemistry based azide-modified proteins on a resin of alkyne agarose. Superior to biotin or lectin based enrichment approaches. Ideal for proteomics, biomarker discovery, posttranslational modification (PTM) analysis, and more. The azide modification can occur via metabolic feeding, enzymatic addition, or chemical modification. Click-Azide modified proteins, or their post-translationally modified forms, are enriched from complex protein extracts on the alkyne-agarose resin supplied. Once anchored to the resin via copper catalyzed click chemistry, extensive washing yields a highly enriched population of nascent molecules that can be furthered characterized by mass spectrometry. The alkyne-agarose resin improves upon existing biotin approaches by>8 fold, with signal to noise of biotin = 3 while the Click-iT resin = 25.

Perfect upstream MS-based sample preparation technique for looking at changes in global protein expression and biomarker analysis.

The new Click-iT® enrichment resin affords many distinct advantages including::

  1. global differential profiling of multiple subclasses of posttranslationally modified (PTM) proteins and newly synthesized proteins


  2. improved signal to noise by eliminating nonspecific binding and increasing selectivity, thus improving detection of low abundant proteins


  3. accelerates PTM site identification leading to more rapid initiation of functional studies

  4. fully compatible with widely used MS techniques including iTRAC and ICAT.


Seamless integration of cell biology with protein chemistry.

Affordable, easy, no special equipment required.

To know what’s new in your cells use Click-iT®

Click-iT®: One Reaction, Endless Possibilities.

5(6)-SFX (6-(Fluorescein-5-(and-6)-Carboxamido) Hexanoic Acid, Succinimidyl Ester), mixed isomers (Invitrogen™)

Fluorescein is a common green-fluorescent derivatization reagent, while succinimidyl esters are frequently used for the labeling of the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. This "SFX" succinimidyl ester contains a seven-atom amino-hexanoyl spacer between the fluorophore and the reactive group. This spacer separates the fluorophore from the biomolecule to which it is conjugated, potentially reducing the quenching that typically occurs upon conjugation. The resulting fluorescein conjugates can be detected with standard FITC or GFP filter sets. Fluorescein-based dyes and their conjugates have a relatively high rate of photobleaching and a pH-sensitive fluorescence. Alexa Fluor® 488 and Oregon Green® 488 are alternative dyes whose spectra mimic those of fluorescein and are much more photostable with little to no pH sensitivity in the physicological pH range.

Specifications:

• Fluorophore label : fluorescein

• Reactive group: succinimidyl ester

• Reactivity: primary amines on proteins and ligands, amine-modified oligonucleotides

• Molecular weight: 586

• Extinction coefficient: 74,000 cm-1M-1

• Ex/Em of the conjugate: 494/520 nm

• Spectrally similar dyes: Alexa Fluor® 488, GFP

Typical Conjugation Reaction

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

The fluorescein succinimidyl ester is typically dissolved in high-quality, anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO) and the reaction 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 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.

Alternative Packagings

Mixed-isomer amine-reactive succinimidyl esters of fluorescein are also available in a 10 x 1 mg unit size (F6129), a single isomer (F6106), and with a spacer that is more hydrophilic than the SFX spacer (F6130), as well as in numerous kits for labeling proteins and nucleic acids (Active esters and kits for labeling proteins and nucleic acids—Table 1.2).

EZ-Link™ NHS-Desthiobiotin (Thermo Scientific™)

Thermo Scientific EZ-Link NHS-Desthiobiotin is a short, membrane permeable, amine-reactive labeling reagent whose biotin-like group is elutable from streptavidin, making it ideal for intracellular protein labeling and purification.

Features of EZ-Link NHS-Desthiobiotin:

Desthiobiotin – analog of biotin allows easy elution from streptavidin, an ideal feature for affinity purification applications
Protein labeling – tag antibodies or proteins in purified or mixed samples to enable high recovery in pull-down assays with streptavidin beads
Membrane-permeable – achieve intracellular labeling with intact cells because the small, uncharged reagent easily diffuses across cell membranes
Amine-reactive – reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the amino-termini of polypeptides

NHS-Desthiobiotin is a variant of biotin that is activated as an NHS ester to covalently label primary amines (-NH2) of proteins or other molecules with desthiobiotin groups. The desthiobiotin tag binds to streptavidin and other biotin-binding proteins with high specificity yet readily elutes with mild conditions (i.e., by competitive displacement with regular, free biotin). As such, this reagent is a useful alternative to NHS-Biotin (Part No. 20217) for avidin-biotin techniques in which nondenaturing elution of the labeled proteins is desired. This simplest and smallest variety of amine-reactive desthiobiotin reagent is uncharged and cell membrane permeable; therefore, NHS-Desthiobiotin can be used with intact cells for intracellular protein labeling.

Desthiobiotin vs. Biotin
Desthiobiotin is a single-ring, sulfur-free analog of biotin that binds to streptavidin with nearly equal specificity but less affinity than biotin (1/Kd = 10^11 vs. 10^15M, respectively)[1-2]. Consequently, desthiobiotinylated bait proteins and their interacting partners can be eluted readily and specifically from streptavidin affinity resin using mild conditions based on competitive displacement with free biotin. For pull-down assay experiments with biological samples, this soft-release characteristic of desthiobiotin also helps to minimize co-purification of endogenous biotinylated molecules, which remain bound to streptavidin upon elution of the target protein complexes with free biotin. The modified avidin-biotin affinity system also eliminates the need to use harsh elution conditions that might disassociate complexes and/or damage the target protein or cell. Desthiobiotin-based techniques are ideal when using native or recombinant proteins that are not expressed with a fusion tag and when isolating captured proteins under native conditions, such as targeting intact cells or cell surface proteins.

Labeling with NHS Ester Reagents
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 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. Most sulfo-NHS esters are directly water soluble but not membrane permeable. Plain NHS esters are less soluble in aqueous buffers but are membrane permeable.

BODIPY™ FL Iodoacetamide (BODIPY™ FL C1-IA, N-(4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-yl)Methyl)Iodoacetamide) (Invitrogen™)

The thiol-reactive BODIPY® FL iodoacetamide can be used to create green-fluorescent bioconjugates. The electronically neutral BODIPY® FL dye has the most fluorescein-like spectra of the green-fluorescent BODIPY® dyes.

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.

DyLight™ 405 NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight 405 Amine-Reactive Dye is an NHS ester-activated derivative of high-performance DyLight 405 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods.

DyLight 405 has a vibrant blue fluorescence intensity that replaces Alexa Fluor™ 405, Cascade™ Blue and AMCA in many applications. The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates. DyLight 405 Amine-Reactive Dye is also available as part of two antibody labeling kit sizes.

Features of the DyLight 405 Amine-Reactive Dye:

High performance— DyLight 405 shows bright fluorescence and replaces Alexa Fluor 405, Cascade Blue and AMCA
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 405 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 405 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 405 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 405 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™ 405 Antibody Labeling Kit
DyLight™ 405 Microscale Antibody Labeling Kit

Texas Red™-X, Succinimidyl Ester, single isomer (Invitrogen™)

The amine-reactive Texas Red®-X, succinimidyl ester can be used to can be used to create bright red-fluorescent bioconjugates with excitation/emission maxima ~595/615 nm. This reactive dye contains an additional seven-atom aminohexanoyl spacer ("X") between the fluorophore and the succinimidyl 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.

Alexa Fluor™ 546 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.

Cascade Blue™ Acetyl Azide, Trisodium Salt (Invitrogen™)

The amine-reactive Cascade Blue® acetyl azide and its conjugates yield blue-fluorescence (approximate excitation/emission maxima ~396/410 nm) that can be used as a contrasting color in multicolor applications. When compared with aminocoumarin derivatives, the sulfonated pyrene Cascade Blue® dye shows less spectral overlap with fluorescein. In addition, Cascade Blue® dye exhibits high absorptivity, is highly fluorescent and, unlike most dyes, resists quenching upon protein conjugation.

NeutrAvidin™ Protein, Maleimide-Activated (Thermo Scientific™)

Thermo Scientific Maleimide-Activated NeutrAvidin Protein is a specially-prepared form of avidin that decreases background in biotin-binding.

Features of NeutrAvidin Protein:

Near-neutral isoelectric point—pI = 6.3, more neutral than native avidin
Nearly devoid of glycosylation—decreased possibility of lectin binding compared to native avidin
No RYD recognition sequence—no known off-target binding domains like streptavidin
Affordable—significantly less expensive than streptavidin

NeutrAvidin Protein is deglycosylated native avidin from egg whites. Removal of the excess carbohydrate by an exclusive process yields a protein with a more neutral isoelectric point and less nonspecific binding properties. Conjugated NeutrAvidin Protein provides exceptional performance in western blot, ELISA and IHC applications that require biotin-binding probes. Assay specificity, sensitivity, and signal-to-noise ratios with NeutrAvidin Protein are generally equivalent or better than with the significantly more expensive streptavidin.

Avidin is a glycoprotein found in the egg white and tissues of birds, reptiles and amphibia. The biotin-binding protein contains four identical subunits having a combined mass of 67,000-68,000 daltons. Removing the glycosylation from avidin yields NeutrAvidin Protein with a mass of 60,000 daltons. Carbohydrate-based lectin-binding is reduced to undetectable levels, yet biotin-binding affinity is retained. NeutrAvidin Protein offers the advantages of a neutral pI to minimize nonspecific adsorption, along with lysine residues that remain available for derivatization or other customized conjugation. NeutrAvidin Protein yields the lowest nonspecific binding among the known biotin binding proteins. The specific activity for biotin-binding is approximately 14 µg/mg of protein, which is near the theoretical maximum activity.

Related Products
NeutrAvidin™ Protein
NeutrAvidin™ Protein, Horseradish Peroxidase Conjugated
NeutrAvidin™ Protein, Alkaline Phosphatase Conjugated
NeutrAvidin™ Protein, Fluorescein Conjugated

Qdot™ 545 ITK™ Amino (PEG) Quantum Dots (Invitrogen™)

Qdot® 545 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® 545 ITK™ amino quantum dot has emission maxima of ~545 nm
• Extremely photostable and bright fluorescence
• Efficiently excited with single-line excitation sources
• Narrow emission, large stokes shift
• Available in multiple colors
• Ideal 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.

Texas Red™ C2 Maleimide (Invitrogen™)

The thiol-reactive Texas Red® C2 maleimide can be used to can be used to create bright red-fluorescent bioconjugates with excitation/emission maxima ~595/615 nm.

DyLight™ 488, Free Acid (Thermo Scientific™)

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

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

General DyLight Fluorophore Highlights:

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

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

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™ 775-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 775-B2 dye has a structure based on the benzopyrillium core, with 2 sulfonates. It has excitation and emission peaks at 772 and 787 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™ 775-B3 NHS Ester
DyLight™ 775-B4 NHS Ester

FluoReporter™ Biotin Quantitation Assay Kit, for biotinylated proteins (Invitrogen™)

The FluoReporter® Biotin Quantitation Assay Kit provides a sensitive fluorometric assay for determining the number of biotin labels on nucleic acid samples (i.e., cDNA). The assay uses Biotective™ Green reagent, which contains a fluorescent dye that is quenched by ligands occupying the biotin binding sites. In the presence of biotin, the quencher dye ligands are displaced and fluorescence occurs. The fluorescence is proportional to the amount of added biotin. The results can be read with any fluorescence-based microplate reader capable of detecting FITC dye.

Important Features of FluoReporter® Biotin Quantitation Assay Kits:
• Can detect from 4 to 80 picomoles of biotin in a sample (50-fold higher sensitivity than HABA biotin-binding assay)
• Can be applied to as little as 13 ng of biotin-labeled nucleic acid (more is needed for lower degrees of labeling)
• Excitation/emission maxima of the reagent is 495/519 nm
• Supplied with a biotinylated positive control for standard curve


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

6-ROX (6-Carboxy-X-Rhodamine), single isomer (Invitrogen™)

The excitation and emission spectra of carboxyrhodamine 6G (CR 6G) fall between those of fluorescein and tetramethylrhodamine. With a peak absorption at 525 nm, conjugates of carboxyrhodamine 6G are an excellent match to the 514 nm spectral line of the argon-ion laser. The amine-reactive 6-CR 6G, SE can be used to attach this fluorophore to biomolecules of interest including amine-modified oligonucleotides.

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

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

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

Protein labeling—biotinylate antibodies to facilitate immobilization, purification or detection using streptavidin resins or probes
Cell surface labeling—biotinylates only surface proteins of whole cells because the negatively charged reagent does not permeate cell membranes
Amine-reactive—reacts with primary amines (-NH2), such as 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
Medium length—spacer arm (total length added to target) is 22.4 angstroms; provides excellent balance between adequate length (minimal steric hindrance for biotin binding) and modest mass

Sulfo-NHS-LC-Biotin is one 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-LC-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-LC-Biotin
EZ-Link™ Sulfo-NHS-LC-Biotinylation Kit

DyLight™ 350 NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight 350 Amine-Reactive Dye is an NHS ester-activated derivative of high-performance DyLight 350 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods.

DyLight 350 is a derivative of aminomethylcoumarine acetate (AMCA) and has a higher fluorescence intensity than Alexa Fluor™ 350 and AMCA in many applications. The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates. DyLight 350 Amine-Reactive Dye is also available as part of two antibody labeling kit sizes.

Features of DyLight 350 NHS Ester:

High performance—DyLight 350 shows brighter fluorescence than Alexa Fluor 350 and AMCA
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 350 Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

Typical labeling reactions require DyLight 350 Amine-Reactive Dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to specific amounts of the labeling reagent. This feature allows DyLight 350 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 350 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 350 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™ 350 Antibody Labeling Kit
DyLight™ 350 Microscale Antibody Labeling Kit

Qtracker™ 800 Cell Labeling Kit (Invitrogen™)

The reagents in the Qtracker® 800 Cell Labeling Kit use a custom targeting peptide to deliver near-infrared-fluorescent Qdot® 800 nanocrystals into the cytoplasm of live cells. Qtracker® Cell Labeling Kits are designed for loading cells grown in culture with highly fluorescent Qdot® nanocrystals. Once inside the cells, Qtracker® labels provide intense, stable fluorescence that can be traced through several generations, and are not transferred to adjacent cells in a population.

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

Key Attributes:

Qtracker® 800 label has Ex/Em (405-760/800) nm
Designed for loading cells grown in culture with highly fluorescent Qdot® nanocrystals
Provide intense, stable fluorescence that can be traced through several generations
Available in eight colors—525 nm, 565 nm, 585 nm, 605 nm, 625 nm, 655 nm, 705 nm, or 800 nm emission
Excellent tools for long-term tracking or imaging studies of live cells, including migration, motility, morphology, and other cell function assays

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

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

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

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

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

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

Rhodamine Green™-X, Succinimidyl Ester, Hydrochloride, mixed isomers (Invitrogen™)

The thiol-reactive Rhodamine Green®-X, succinimidyl ester can be used to create green-fluorescent bioconjugates with excitation/emission maxima ~502/527 nm. This dye is the nonsulfonated analog of the Alexa Fluor® 488 dye.

Alexa Fluor™ 350 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 blue fluorescent Alexa Fluor® 350. 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® 350 Antibody Labeling Kit:
• With an excitation and emission maximum of 346/442 nm, Alexa Fluor® 350 can be efficiently excited using a UV laser line and detected under standard DAPI 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.

Lissamine™ Rhodamine B Ethylenediamine (Invitrogen™)

The red-fluorescent Lissamine™ rhodamine B ethylenediamine 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).

Texas Red™ Cadaverine (Texas Red™ C5) (Invitrogen™)

The primary aliphatic amine of the red-fluorescent Texas Red® cadaverine (Texas Red® C5) 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).

Alexa Fluor™ 700 NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor® 700 is a bright and photostable near-IR dye that can be excited with a xenon-arc lamp, far-red diode lasers, or dye pumped lasers operating in the 675–700 nm range. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 700 dye is water soluble and pH-insensitive from pH 4 to pH 10. Fluorescence of this long-wavelength Alexa Fluor® dye is not visible to the human eye but is readily detected by most imaging systems. In addition to reactive dye formulations, we offer Alexa Fluor® 700 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection.The NHS ester (or succinimidyl ester) of Alexa Fluor® 700 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® 700 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 702/723 nm
Extinction coefficient: 205,000 cm-1M-1
Molecular weight: ~1400

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.

Zenon™ Alexa Fluor™ 532 Rabbit IgG Labeling Kit (Invitrogen™)

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

Important Features of Zenon® Labeling Technology:

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


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

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

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

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

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

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

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

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

Related Links:

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

Zenon™ Allophycocyanin Mouse IgG1 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.

DTNB; Ellman's Reagent, 5,5'-Dithiobis-(2-Nitrobenzoic Acid) (Invitrogen™)

DTNB or Elman's reagent can be used to quantitate thiols in proteins, cells and plasma by absorption measurements. It readily forms a mixed disulfide with thiols, liberating the chromophore 5-merapto-2-nitrobenzoic acid (absorption maximum 410 nm). Only protein thiols that are accessible to this water-soluble reagent are modified.

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

DyLight™ 550 Antibody Labeling Kit (Thermo Scientific™)

The Thermo Scientific DyLight 550 Antibody Labeling Kit contains an NHS ester-activated derivative of high-performance DyLight 550 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods. The standard size kit contains all necessary components to perform three separate labeling reactions using 1 mg of IgG or similar quantities of other proteins.

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

Features of DyLight 550 NHS Ester:

High performance—DyLight 550 shows brighter fluorescence than Alexa Fluor 555, TRITC and Cy3 dye
Specific—NHS ester-activated dye labels proteins and other molecules at primary amines (-NH2)
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 550 Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

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

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EZ-Link™ NHS-PEG Solid-Phase Biotinylation Kit - 1mL Column (Thermo Scientific™)

Thermo Scientific EZ-Link NHS-PEG Solid Phase Biotinylation Kits provide an on-column approach to amine-targeted antibody biotinylation that provides greater control over the labeling reaction and clean-up process.

Features of the EZ-Link NHS-PEG Solid-Phase Biotinylation Kit:

Ideal for small-scale reactions—0.1 to 1 mg antibody
Fast labeling and purification—the entire procedure takes only about one hour
Easy removal of spent and excess labeling reagent—simply wash away the reaction byproducts—no need for dialysis or gel filtration
No dilution effects—solid-phase method allows initially dilute antibodies to be recovered in a smaller volume after labeling
Optimized protocols—specific protocols for antibody ensure appropriate level of labeling (2 to 5 biotins per antibody molecule), minimizing possibility of inactivation caused by overlabeling
High-performance biotin reagent—NHS-PEG4-Biotin uses efficient, amine-specific NHS-ester chemistry and includes hydrophilic polyethylene glycol (PEG) spacer that stabilizes long-term solubility of labeled antibodies
Eight biotin-labeling experiments per kit—Thermo Scientific No-Weigh Packaging ensures that the biotin reagent is fully active for eight separate experiments

This innovative antibody-labeling system uses nickel-chelated agarose to temporarily immobilize antibody molecules via their histidine-rich Fc regions. Once held in place on the resin, the antibody can be biotinylated at primary amines with the high-quality, solubility-stabilizing NHS-PEG4-Biotin reagent. Excess labeling reagent and byproducts are then washed away before recovering the labeled and purified antibody from the resin using a mild imidazole solution. No gel filtration or dialysis is needed.

EZ-Link Solid-Phase Biotinylation Kits use an innovative strategy for antibody biotinylation that simplifies the process in many respects, making successful biotinylation possible for the novice and expert. The strategy involves immobilizing the antibody to a metal-chelated affinity support and performing the biotinylation in the solid phase.

Antibodies bind readily to metal-chelated supports, such as nickel or cobalt, binding primarily through the histidine-rich Fc region of IgG-class antibodies. This feature allows the derivatization chemistry to be performed easily while the antibody is bound to the support. This strategy facilitates reagent delivery and removal of spent reagent. The resulting modified antibody is then selectively removed from the support by elution with a buffered imidazole solution.

The solid-phase approach to antibody biotinylation provides more control over the reaction. For example: Reagent and antibody ratios and reaction times can be adjusted to achieve the proper degree of biotinylation for the intended application.

The kits contain an aqueous soluble amino group-specific reagent (NHS-PEG4-Biotin) that targets accessible amino (-NH2) groups on the surface of the antibody. The format can be applied at the multi-milligram antibody level in the 1 mL column kit (Part No. 21440) or to low microgram antibody levels for biotinylations using the mini-spin column kit (Part No. 21450).

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FluoReporter™ Mini-Biotin-XX Protein Labeling Kit (Invitrogen™)

The FluoReporter® Mini-biotin-XX Protein Labeling Kit provides a convenient and flexible means for attaching biotin-XX to your antibody or protein (>30 kDa). This kit contains a water-soluble biotin-XX sulfosuccinimidyl ester, where "XX" is a 14-atom spacer that separates the reactive ester from the biotin moiety, making the biotin more accessible to avidin or streptavidin. The kit supplies the reagents needed for 5 to 10 labeling and separation reactions.

Important Features of FluoReporter® Protein Labeling Kits:
• Labeled proteins typically ready to use in under 3 hours with very little hands-on time
• Optimized for 0.1–0.6 mg of protein with molecular weight >30 kDa
• Labeled proteins are purified using convenient spin filters
• Requires the antibody or protein be purified prior to labeling


Learn More About Protein Labeling
We offer a wide selection of Molecular Probes® protein and antibody 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 Labeled Protein for You
If you can’t find what you’re looking for in our stocked list, we’ll prepare a custom labeled protein for you. Our custom services are 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 human or animal therapeutic or diagnostic use.

Pierce™ Fluorescence Biotin Quantitation Kit (Thermo Scientific™)

Thermo Scientific Pierce Fluorescence Biotin Quantitation Kit requires only ten microliters of sample to accurately measure the biotinylation level of labeled antibodies and other biomolecules.

Features of the Fluorescence Biotin Quantitation Kit:

Fast—requires only 5 minute incubation
Economical—requires only 10 µL of sample (approx. 750ng of biotinylated IgG)
Sensitive—linear working range of 10 to 60 picomoles of biotin

This microplate-based biotin assay is easy to perform by adding the supplied fluorescent reporter to the biotinylated samples and diluted biocytin standards. The avidin fluoresces when the weakly interacting HABA (4'-hydroxyazobenzene-2-carboxylic acid) is displaced by the biotin. The amount of biotin is determined by comparing the sample's fluorescence to the biocytin standard curve. This assay requires must less sample volume than the microplate colorimetric HABA assay and is much more sensitive.

Includes
Assay buffer, HABA-avidin fluorescent reporter solution, and biocytin control

Requires
Black opaque 96-well microplate and a fluorescence plate reader (excitation 494 nm; emission 520 nm)

The highly specific interaction of avidin or streptavidin with biotin is the basis for many purification and detection systems. Quantifying the degree of biotinylation is necessary for assessing if a biotin-labeling procedure was successful and what amount of biotinylated molecule to use for a specific application.

HABA (4'-hydroxyazobenzene-2-carboxylic acid) is a dye that weakly interacts with avidin and is commonly used in a colorimetric assay to quickly estimate the biotin-to-protein ratio. The Fluorescence Biotin Quantitation Kit also uses HABA dye but is more sensitive and accurate. A premix of fluorescent avidin with HABA (DyLight Reporter) is added to the solution containing the biotinylated sample. Because of its higher affinity for avidin, biotin displaces the HABA, allowing the avidin to fluoresce. The amount of biotin is measured in a microplate by comparing the fluorescence to a biocytin standard curve.

The Fluorescence Biotin Quantitation Kit is requires only 10 µL of sample. By contrast, the traditional colorimetric method requires 100 µL of sample, and another leading supplier's fluorescent assay kit requires 50 µL of sample. Our kit is more accurate than the other supplier's fluorescent biotin assay (Brand I).

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

Thermo Scientific EZ-Link Hydrazide-Biotin is the shortest and simplest hydrazide-activated biotinylation reagent for labeling glycoproteins and other carbohydrate-containing compounds having oxidizable sugars or aldehydes.

Features of EZ-Link Hydrazide-Biotin:

Glycoprotein labeling—biotinylate glycosylated proteins at sialic acid residues for detection or purification using streptavidin probes or resins
Cell surface labeling—biotinylate and isolate cell surface glycoproteins
Aldehyde-reactive—reacts with aldehydes formed by periodate-oxidation of sugar groups
Hydrazide-activated—perform reactions at pH 4 to 6 in buffers such as sodium acetate
Irreversible—forms semi-permanent hydrazone bonds; spacer arm cannot be cleaved
Solubility—usually dissolved in DMSO before further dilution in aqueous buffers
Spacer arm length—15.7Å

This biotin hydrazide reagent enables simple and efficient biotin labeling of polyclonal antibodies and other glycoproteins. Mild oxidation of antibodies with sodium periodate produces reactive aldehydes on the carbohydrate moieties of the Fc portion that can be modified by hydrazides. This approach is advantageous for labeling antibodies because biotinylation occurs only at the sites of glycosylation, which are primarily in the Fc region of the antibody, far from the antigen binding site.

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. Hydrazides and alkoxyamines are two types of carbonyl-reactive groups. Hydrazides (—NH-NH2) react specifically with aldehyde groups in slightly acidic conditions to form hydrazone linkages; these can be further reduced to stable secondary amine bonds using sodium cyanoborohydride (Part No. 44892). The reaction is more efficient in the presence of aniline (Part No. 88944). Alternatively, hydrazides can be conjugated to carboxylic acids using EDC carbodiimide chemistry.

Reactive aldehyde groups can be generated in glycoproteins and other polysaccharide compounds by oxidation of constituent sugar diols using sodium periodiate (Part No. 20504). Sialic acid residues are common components of protein glycosylation and are easily converted to aldehydes with 1 mM NaIO4.

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

Thermo Scientific Pierce Plus Activated Peroxidase is an amine-reactive form of horseradish peroxidase (HRP) that provides coupling efficiencies of greater than 95% with antibodies and other proteins. Each kit contains 5 x 1 mg of lyophilized HRP and sufficient Sodium cyanoborohydride solution, quenching buffer, and buffers to conjugate 5 mg of Immunoglobulin (IgG)

Features of the Thermo Scientific Pierce Plus Activated Peroxidase Kit:

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

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

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