3D image of protein and chemical structure

Protein labeling reagents are reactive dyes that attach to a specific functional group (i.e., amine or thiol) on a target biomolecule allowing for its subsequent detection or purification.

Use the Protein Labeling Reagents Selection Tool to find the best reactive dye for your application or learn more about the different reactive dyes below. Additionally, access our bioconjugation technical handbook to help improve your bioconjugation results.

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Protein labeling reagents not only bind proteins but other molecules including antibodies, peptides, ligands, and synthetic oligonucleotides. They are offered for use with various applications such as immunochemistry, fluorescence in situ hybridization (FISH), and receptor labeling, as well as for probing biological structure, function, and interactions.

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Amine labeling

Amine reactive dyes are used to label proteins, peptides, ligands, synthetic oligonucleotides, and other biomolecules. Most commonly, they are used to prepare bioconjugates for immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), cell tracing, receptor labeling, and fluorescent analog cytochemistry. During these processes, the stability of the chemical bond between dye and biomolecule is critical since the probe will often be subjected to a series of post-processing steps such as washing, permeabilization, fixation, and mounting.

There are three major classes of reagents used to label amines: active esters, isothiocyanates (ITC), and sulfonyl chlorides (SC). Active esters such as succinimidyl esters (commonly known as NHS esters), tetrafluorophenyl esters (TFP), and sulfodichlorophenol esters (SDP) are preferred for the conjugation to proteins because they form a very stable amide bond between the dye or hapten and the protein (Figure 1). Learn more about amine-reactive derivatives.

Label an amine-reactive molecule using our Protein Labeling Reagent Selection Tool

Reaction of amine reactive group with succinimidyl ester or TFP ester fluorescent dye to form a carboxamide conjugate
Figure 1. Reaction of a primary amine with a succinimidyl ester or a tetrafluorophenyl (TFP) ester.

Thiol labeling

Thiol-reactive dyes, also known as sulfhydryl-reactive, are principally used to label fluorescent proteins, peptides, and sulfhydryl-modified oligos for analyzing protein structure and function. Thiol-reactive reagents include iodoacetamides, maleimides, benzylic halides and bromomethylketones, which react by S-alkylation of thiols to generate stable thioether products. Iodoacetamides readily react with thiols found on peptides, proteins, and thiolated polynucleotides. While maleimides are optimal for thiol-selective modification, quantitation, and analysis where the thiol is added across the double bond of the maleimide (Figure 2). Learn more about thiol reactive probes

Label a thiol-reactive molecule using our Protein Labeling Reagent Selection Tool

Reaction of thiol reactive group with a maleimide fluorescent dye to form a thioester conjugate
Figure 2. Reaction of a thiol with a maleimide.

Carbonyl labeling

A carbonyl group is composed of a carbon double-bonded to an oxygen (C=O). Types of carbonyl groups include aldehydes, ketons, and carboxylic acids. Learn more about modifying carbonyl functional groups

Hydrazide and hydroxylamine derivatives
Hydrazine and hydroxylamine derivatives are available for reaction with aldehydes and ketones in low molecular weight structures such as drugs, steroid hormones, reducing sugars, and metabolic intermediates.
ProductChemical reactivity on moleculeReactive moiety on dyeEx/EmCat. No.
Alexa Fluor 488 Hydrazidealdehydes and ketonesHydrazine493/517A10436
Alexa Fluor 594 Hydrazidealdehydes and ketonesHydrazine588/613A10438
Alexa Fluor 633 Hydrazidealdehydes and ketonesHydrazine624/643A30634
Alexa Fluor 647 Hydrazidealdehydes and ketonesHydrazine649/666A20502
Alexa Fluor 488 Cadaverinecarboxylic acid, aldehydes, ketonesAmine493/516A30676
Alexa Fluor 555 Cadaverinecarboxylic acid, aldehydes, ketonesAmine555/572A30677
Alexa Fluor 647 Cadaverinecarboxylic acid, aldehydes, ketonesAmine651/672A30679
Carboxylic acids
Carboxylic acids are non-reactive derivatives that can be used as reference standards for dye-conjugates or converted to esters, amides, acyl hydrazides, or hydroxamic acids.
ProductChemical reactivity on moleculeReactive moiety on dyeEx/EmCat. No.
Alexa Fluor 350 Carboxylic Acid, bis(triethylammonium) saltAmineCarboxylic acid346/442A33076
Alexa Fluor 488 Carboxylic Acid, tris(triethylammonium) saltAmineCarboxylic acid500/525A33077
Alexa Fluor 532 Carboxylic Acid, triethylammonium saltAmineCarboxylic acid530/554A33078
Alexa Fluor 546 Carboxylic Acid, tris(triethylammonium) saltAmineCarboxylic acid556/573A33079
Alexa Fluor 555 Carboxylic Acid, tris(triethylammonium) saltAmineCarboxylic acid555/580A33080
Alexa Fluor 568 Carboxylic Acid, tris(triethylammonium) saltAmineCarboxylic acid578/603A33081
Alexa Fluor 594 Carboxylic Acid, tris(triethylammonium) saltAmineCarboxylic acid590/617A33082
Alexa Fluor 633 Carboxylic Acid, tris(triethylammonium) saltAmineCarboxylic acid632/647A33083
Alexa Fluor 647 Carboxylic Acid, tris(triethylammonium) saltAmineCarboxylic acid650/665A33084
Alexa Fluor 750 Carboxylic Acid, tris(triethylammonium) saltAmineCarboxylic acid749/775A33085
BODIPY FL (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Propionic Acid)AmineCarboxylic acid505/509D2183
DyLight 488 Free AcidAmineCarboxylic acid593/51862280
6-FAM (6-Carboxyfluorescein), single isomerAmineCarboxylic acid492/514C1360
5(6)-TAMRA (5-(and-6)-Carboxytetramethylrhodamine), mixed isomersAmineCarboxylic acid543/570C300
5-TAMRA (5-Carboxytetramethylrhodamine), single isomerAmineCarboxylic acid541/570C6121
5-ROX (5-Carboxy-X-Rhodamine, Triethylammonium Salt), single isomerAmineCarboxylic acid578/604C6124
6-ROX (6-Carboxy-X-Rhodamine), single isomerAmineCarboxylic acid577/589C6156

Click chemistry

Click chemistry is a powerful method for quickly and specifically joining biomolecules. It describes a class of chemical reactions that use bio-orthogonal or biologically unique moieties to label and detect a molecule of interest. The small click label can easily penetrate samples, and the selectivity and stability of the reaction makes it highly sensitive with low background signal. Click chemistry is an ideal detection method for samples that may be compromised by direct labeling or antibody-based secondary detection techniques.

Alkyne and azide labeling

Click chemistry is a two-step process that uses a copper-catalyzed triazole formation from an azide and an alkyne (Figure 3). The azide and alkyne moieties can be used interchangeably; either one can be used to tag the molecule of interest, while the other is used for subsequent detection.

Label an alkyne- or azide reactive molecule using our Protein Labeling Reagent Selection Tool

Low-copper reactions

Picolyl azides allow copper-catalyzed click reactions with copper sensitive compounds, all while retaining the benefits of the original copper-catalyzed reactions. Some biomolecules, such as GFP and RPE, can exhibit reduced fluorescence and nucleic acids can be denatured by high-copper environments. These kits are optimized for use with proteins (e.g., GFP, RPE), nucleic acids (e.g., RNA, oligos), and small molecules (e.g., phalloidin). These reactions are ideal for intracellular staining.

Copper-free reactions

Click-iT DIBO and sDIBO alkynes label azide-containing molecules via a copper-free click reaction. Detection has similar sensitivity to the copper-catalyzed click reaction without the use for copper catalysis. sDIBO yields less ‘sticky’ conjugates and lower background signal in samples versus DIBO. Copper-free click reactions allow for in vivo and in vitro detection and labeling of nucleic acids, proteins, peptides, and other small molecules for monitoring biological viability and activity. These reactions are ideal for surface labeling and minimize damage to fluorescent proteins such as GFP or RPE.

 Learn more about Click-iT and Click-iT DIBO detection assays—Green Fact Sheet

Buffers for standard Click-iT reactions

Click-iT Reaction Buffer Kits are optimized for proteins or cell samples that are labeled with an azide- or alkyne-tagged biomolecule.

Click-iT Cell Reaction Buffer KitPerform 50 reactions based on a 0.5 mL reaction volume for subsequent analysis for flow cytometry, fluorescence microscopy, or high-content screening (HCS)
Click-iT Protein Reaction Buffer KitPerform the click reaction of proteins for subsequent standard protein biochemical analysis (e.g., western blots, mass spectrometry).

Click-iT Assays

The Click-iT technology has a streamlined and simplified workflow for a variety of applications including imaging, flow cytometry, and high-content analysis (HCA). Several optimized assay kits have been developed that measure cell proliferation, protein synthesis, RNA synthesis, lipid peroxidation, and apoptosis. The sensitive Click-iT technology can also be used for molecular capture of nascent proteins and RNA. Find specific click chemistry products below.

Click-iT EdU assays
Combine EdU labeling with click chemistry to provide a superior alternative to traditional BrdU staining methods for detecting and quantitating newly synthesized DNA.

Bivariant plot with replicating CD3 positive cells in upper right and non-replicating CD3 positive cells in upper left
Immunophenotyping experiment to evaluate CD3 and DNA strand breaks in human T cells. Dual parameter plot of Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit and Hu CD3 PE-Cy7 fluorescence.

Protein synthesis assays
Utilize the Click-iT Plus technology to label nascent proteins with a range of fluorescent labels, and the opportunity for temporal studies of synthesis and degradation using pulse-chase-type experiments. Additionally, the Click-iT technology can be used to study post-translational modifications including fatty acid attachment and glycosylation.

Sample showing purple glycoproteins and green DNA
Post-translational modification using Click-iT glycoprotein reagent. Tetrahymena pyriformis staining using Click-iT GalNAz glycoprotein labeling reagent. Following fixation and permeabilization using the Image-iT Fixation/Permeabilization Kit, EdU-incorporated DNA was labeled with Alexa Fluor 488 azide and GalNAz-incorporated cellular components with Alexa Fluor 555 alkyne.

Click-iT TUNEL assays
Incorporate EdUTP (a dUTP modified with a small, bioorthogonal alkyne moiety) at the 3’-OH ends of fragmented DNA by the TdT enzyme, for the detection of apoptosis using fluorescence- or colorimetric-based strategies.

apoptotic cells with green caspase-3, orange tubulin, blue nuclei, and light blue overlay of caspase, nuclei, and magenta TUNEL
Late-stage apoptosis visualized using the Click-iT TUNEL Imaging Assay. HeLa cells were treated with staurosporine, then fixed and permeabilized. The Click-iT TUNEL Alexa Fluor 647 Imaging Assay was performed. Activated caspase-3 was detected with a rabbit polyclonal primary antibody for cleaved caspase-3 and labeled with Alexa Fluor 488 goat anti-rabbit IgG antibody (green). Tubulin was detected with a mouse monoclonal anti-tubulin antibody and labeled with Alexa Fluor 555 goat anti-mouse IgG (orange). Nuclei were stained with Hoechst 33342 (blue). The light blue color represents an overlay of caspase (green), Hoechst (blue), and TUNEL (magenta) signals.

Nucleic acid labeling
Use click chemistry tools to either label or capture nascent DNA or RNA for subsequent detection or analysis.

EU incorporation shows red RNA, green tubulin, and blue nuclei
Multiplex imaging with Click-iT RNA assays. NIH3T3 cells were incubated with 1 mM EU for 1 hr, then fixed and permeabilized. EU incorporated into newly synthesized RNA (red) in some cells was detected using the Click-iT RNA Alexa Fluor 594 Imaging Kit. Tubulin (green) was detected with mouse anti-tubulin IgG and visualized with Alexa Fluor 488 Goat Anti-Mouse IgG. Nuclei (blue) were stained with Hoechst 33342.

For Research Use Only. Not for use in diagnostic procedures.