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.
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.
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
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
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.|
|Product||Chemical reactivity on molecule||Reactive moiety on dye||Ex/Em||Cat. No.|
|Alexa Fluor 488 Hydrazide||aldehydes and ketones||Hydrazine||493/517||A10436|
|Alexa Fluor 594 Hydrazide||aldehydes and ketones||Hydrazine||588/613||A10438|
|Alexa Fluor 633 Hydrazide||aldehydes and ketones||Hydrazine||624/643||A30634|
|Alexa Fluor 647 Hydrazide||aldehydes and ketones||Hydrazine||649/666||A20502|
|Alexa Fluor 488 Cadaverine||carboxylic acid, aldehydes, ketones||Amine||493/516||A30676|
|Alexa Fluor 555 Cadaverine||carboxylic acid, aldehydes, ketones||Amine||555/572||A30677|
|Alexa Fluor 647 Cadaverine||carboxylic acid, aldehydes, ketones||Amine||651/672||A30679|
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.
Nobel Prize-winning click chemistry: it’s origins and future impact
The Nobel Prize in Chemistry 2022 has been awarded to three scientists for their pioneering work in click chemistry, a revolutionary technology with significant impacts on various fields of science and medicine. Learn more about click chemistry and its implications in this article by the New England Journal of Medicine.
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
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.
|Alexa Fluor 488||Click-iT Plus Alexa Fluor 488 picolyl azide toolkit (includes reaction buffers)||Picolyl azide||C10641|
|Alexa Fluor 555||Click-iT Plus Alexa Fluor 555 picolyl azide toolkit (includes reaction buffers)||Picolyl azide||C10642|
|Alexa Fluor 647||Click-iT Plus Alexa Fluor 647 picolyl azide toolkit (includes reaction buffers)||Picolyl azide||C10643|
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.
|Alexa Fluor 488||Click-iT Alexa Fluor 488 sDIBO alkyne for copper free click chemistry||Alkyne/sDIBO||C20020|
|Alexa Fluor 555||Click-iT Alexa Fluor 555 sDIBO alkyne for copper free click chemistry||Alkyne/sDIBO||C20021|
|Alexa Fluor 594||Click-iT Alexa Fluor 594 DIBO alkyne for copper free click chemistry||Alkyne/sDIBO||C10407|
|Alexa Fluor 647||Click-iT Alexa Fluor 647 sDIBO alkyne for copper free click chemistry||Alkyne/sDIBO||C20022|
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 Kit||Perform 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 Kit||Perform the click reaction of proteins for subsequent standard protein biochemical analysis (e.g., western blots, mass spectrometry).|
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.
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.
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.
Nucleic acid labeling
Use click chemistry tools to either label or capture nascent DNA or RNA for subsequent detection or analysis.
Molecular Probes Handbook
BioProbes Journal articles
For Research Use Only. Not for use in diagnostic procedures.