Labeling and Detection Information

Learn about strategies and techniques for labeling proteins and antibodies for cell biology research. Topics include traditional reactive chemistries like amine and thiol labeling, protein-protein crosslinking, general protein crosslinkers and more. Newer chemistries for labeling proteins include click chemistry based protein and carbohydrate labeling reactions.

Labeling and detection features

On-demand webinar

A practical approach to antibody labeling

The growing number of fluorophores available makes labeling your own antibodies a tempting proposition, but with many antibody labeling solutions available, selecting the best option can be a daunting task.

Protein methods article

Overview of protein labeling

Labeling strategies result in the covalent attachment of different molecules, including biotin, reporter enzymes, fluorophores and radioactive isotopes, to the target protein or nucleotide sequence. While multiple types of labels are available, their varied uses are preferable for specific applications. 


Labeling and detection subtopics


Labeling and detection learning resources

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Type Title Categories
Application note (2015) Quantitation of proliferating cells with the EVOS FL Auto Imaging System antibodies, antibody labeling, ArrayScan, cell proliferation, EVOS FL Auto Microscope, fluorescence microscopy/fluorescence imaging, fluorescent dyes, high content analysis, onstage incubator, phagocytosis
BioProbes article (2006) Invitrogen flow cytometry—building choice  cell culture, cell cycle, cell health, cell isolation, cell signaling, flow cytometer/flow cytometry, fluorescent dyes, Gibco, fluorescence microscopy/fluorescence imaging, nanocrystals, protein labeling, Qdot, Zenon
BioProbes article (2006) Multicolor immunodetection using the violet laser  Alexa Fluor, antibody labeling, flow cytometer/flow cytometry, fluorescent dyes, immunophenotyping, multicolor flow cytometry, nanocrystals, Qdot
BioProbes article (2006) Products for neurobiology research  enzyme activity detection, flow cytometer/flow cytometry, fluorescent dyes, fluorescence microscopy/fluorescence imaging, in situhybridization, nanocrystals, protein detection, protein labeling, Qdot, viability, Zenon
BioProbes article (2006) A guide to antibody labeling and detection  Alexa Fluor, antibody labeling, flow cytometer/flow cytometry, fluorescent dyes, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), multicolor flow cytometry, nanocrystals, Qdot, Zenon
BioProbes article (2006) Develop your own fluorescence-based ELISA  ELISA, antibody labeling,fluorescent dyes, protein quantitation
BioProbes article (2007) Mercator multiplex phosphoantibody-based array  antibody labeling, flow cytometer/flow cytometry, fluorescent dyes, high content analysis, protein labeling, protein quantitation
BioProbes article (2007) The TC-FlAsH TC-ReAsH protein detection choice  fluorescent dyes, fluorescence microscopy/fluorescence imaging, live-cell imaging, protein detection, protein labeling, stem cell research
BioProbes article (2007) O-GlcNAc: Hiding in plain sight  Click-iT, fluorescent dyes, fluorescence microscopy/fluorescence imaging, protein detection, protein labeling, western blotting
BioProbes article (2007) Quantum dots as replacements for tandem dyes in flow cytometry  flow cytometer/flow cytometry, fluorescent dyes, multicolor flow cytometry, nanocrystals, protein labeling, Qdot
BioProbes article (2007) Effortless protein conjugations for small animal in vivo imaging  Alexa Fluor, antibody labeling, fluorescent dyes, fluorescence microscopy/fluorescence imaging, in vivo imaging
BioProbes article (2007) Important additions to the Alexa Fluor dye series  Alexa Fluor, flow cytometer/flow cytometry, fluorescent dyes, fluorescence microscopy/fluorescence imaging, protein labeling, sample stability
BioProbes article (2007) The latest in stem cell research  antibodies, antibody labeling, cell differentiation, cell expansion, flow cytometer/flow cytometry, fluorescent dyes, growth factors, fluorescence microscopy/fluorescence imaging, stem cell research
BioProbes article (2007) Simultaneously visualize surface and intracellular markers  antibody labeling, cell fixatives, flow cytometer/flow cytometry, fluorescent dyes, sample preparation, Zenon
BioProbes article (2007) Mitochondria and cardiac disorders  antibodies, antibody labeling, cardiovascular system, cell structure-mitochondria, fluorescent dyes, fluorescence microscopy/fluorescence imaging, oxidative stress, western blotting
BioProbes article (2007) The use of Qdot nanocrystals in flow cytometry  flow cytometer/flow cytometry, fluorescent dyes, multicolor flow cytometry, nanocrystals, protein labeling, Qdot, viability
BioProbes article (2007) TC-FlAsH and TC-ReAsH reagents  fluorescent dyes, fluorescence microscopy/fluorescence imaging, live-cell imaging, protein labeling
BioProbes article (2007) Streptavidin conjugates of R-phycoerythrin—detection workhorses  flow cytometer/flow cytometry, fluorescent proteins, Luminex, microarrays, multiplex ELISA/Luminex, particles, protein labeling
BioProbes article (2007) Optimally labeled fluorescent antibodies for in vivo animal imaging  Alexa Fluor, antibody labeling, fluorescent dyes, fluorescence microscopy/fluorescence imaging, in vivo imaging
BioProbes article (2007) Click. Discover. Publish  Click-iT, fluorescent dyes, fluorescence microscopy/fluorescence imaging, protein detection, protein labeling, western blotting
BioProbes article (2008) IgG antibody labeling from A to Z—Alexa Fluor to Zenon IgG Antibody Labeling Kits for imaging and flow cytometry  Alexa Fluor, antibody labeling, flow cytometer/flow cytometry, fluorescent dyes, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), protein labeling, Zenon
BioProbes article (2008) Analyzing signaling events with phosphorylation site–specific antibodies—Insights into functional proteomics  antibodies, antibody labeling, cell signaling, fluorescent dyes, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), protein labeling
BioProbes article (2009) A fluorogenic, target-specific reagent for small animal in vivo imaging  Alexa Fluor, antibody labeling, cancer, flow cytometer/flow cytometry, fluorescent dyes, fluorescence microscopy/fluorescence imaging, in vivo imaging, protein labeling, sample preparation
BioProbes article (2009) Is it time to go direct? IgG antibody labeling methods as versatile as your imagination  Alexa Fluor, antibody labeling, flow cytometer/flow cytometry, fluorescent dyes, immunocytochemistry (ICC), immunohistochemistry (IHC), nanocrystals, Qdot, sample preparation, Zenon
BioProbes article (2009) Novel solid-phase antibody labeling  Alexa Fluor, antibody labeling, flow cytometer/flow cytometry, fluorescent dyes, fluorescence microscopy/fluorescence imaging, sample preparation, Zenon
BioProbes article (2010) Fast and efficient solid-phase antibody labeling antibodies, antibody labeling 
BioProbes article (2010) Fluorescent dye labeling made simple antibody labeling, fluorescent dyes, labeling, protein labeling
BioProbes article (2011) A practical guide to immunocytochemistry—Optimized antibody labeling and detection antibodies, antibody labeling 
BioProbes article (2011) New simplified workflow options for maximum-yield protein conjugations—From APEX to Zenon: Molecular Probes protein labeling kits Alexa Fluor, antibodies, antibody labeling, flow cytometry/flow cytometer, immunofluorescence (IF), protein labeling, Zenon
BioProbes article (2012) Automating cell biology—Create your own HCS assay using trusted Molecular Probes dyes antibodies, antibody labeling, cell structure, cell structure-actin, cell structure-lysosomes, cell structure-mitochondria, CellLight, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, high content analysis
BioProbes article (2012) A new application for pHrodo dextrans antibodies, antibody labeling, Click-iT, flow cytometer/flow cytometry, dextrans, fluorescence particles, protein labeling, Qdot
BioProbes article (2015) Applications for therapeutic monoclonal antibodies—Tools for studying antibody internalization and trafficking antibodies, antibody-drug conjugates, antibody labeling, drug research, endocytosis/phagocytosis, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), immunohistochemistry (IHC), internalization, pH detection
BioProbes article (2017) Double vision: Simultaneous visualization of protein and RNA targets—ViewRNA Cell Plus Assay for antibody labeling and in situ hybridization in individual cells antibody labeling, fluorescence microscopy/fluorescence imaging, fluorescent dyes, in situ hybridization,ViewRNA
BioProbes article (2017) Effectively monitor antibody internalization and trafficking—Improved labeling tools for antibody–drug conjugate screening and characterization antibodies, antibody-drug conjugates, antibody labeling, drug research, endocytosis/phagocytosis, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), immunohistochemistry (IHC), internalization, pH detection
BioProbes article (2017) Amplify signals for fluorescence imaging—SuperBoost kits with Alexa Fluor tyramides for signal amplification Alexa Fluor, antibody labeling, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), secondary detection, signal amplification
Molecular Probes Handbook Phycobiliproteins—Section 6.4 antibody labeling, flow cytometer/flow cytometry, multicolor flow cytometry
Molecular Probes Handbook Thiol-reactive probes excited with visible light—Section 2.2 protein labeling, fluorescent dyes
Molecular Probes Handbook Thiol-reactive probes excited with ultraviolet light—Section 2.3 protein labeling, fluorescent dyes
Molecular Probes Handbook Introduction to thiol modification and detection—Section 2.1 antibody labeling, protein labeling, sulfhydryl modification, thiol labeling, thiol modification
Molecular Probes Handbook Labeling small peptides with amine-reactive dyes in organic solvents—Note 9.2 amine-reactive dyes, fluorescence, peptide labeling, protein modification
Molecular Probes Handbook Derivatization reagents for carboxylic acids and carboxamides—Section 3.4 amine-reactive dyes, amines, carboxylic acid modification, fluorescence, hydroxylamines, labeling chemistries, protein modification and crosslinking
Molecular Probes Handbook Click chemistry—Section 3.1 azide and alkyne labeling reagents, azide–alkyne cyloaddition, bioorthogonal labeling, BrdU alternative, click reaction, Click-iT, EdU
Molecular Probes Handbook Reagents for analysis of low molecular weight amines—Section 1.8 amine labeling, ATTO-TAG Reagents, dialdehydes: OPA and NDA, fluorescamine, protein modification
Molecular Probes Handbook Kits for labeling proteins and nucleic acids—Section 1.2 antibody labeling, nucleic acid labeling, protein labeling, protein modification
Molecular Probes Handbook Introduction to amine modification—Section 1.1 amine labeling, amine modification, antibody labeling, protein labeling, protein modification
Molecular Probes Handbook Introduction to avidin–biotin and antibody–hapten techniques—Section 4.1 antibodies, avidins and streptavidins, fluorescence, labeling and detection, secondary antibodies, secondary detection, signal amplification
Molecular Probes Handbook Biotinylation and haptenylation reagents—Section 4.2 antibody labeling, biotinylation, protein labeling, secondary detection, signal amplification
Molecular Probes Handbook Biotin and desthiobiotin conjugates—Section 4.3 antibody labeling, biotinylation, protein labeling, secondary detection, signal amplification
Molecular Probes Handbook Zenon Technology: Versatile reagents for immunolabeling—Section 7.3 antibody labeling, flow cytometer/flow cytometry, fluorescence microscopy/fluorescence imaging, secondary detection, Zenon
Molecular Probes Handbook Introduction to antibodies, avidins and lectins—Section 7.1 antibodies, avidins and streptavidins, fluorescence, labeling and detection, secondary antibodies, secondary detection, signal amplification
Molecular Probes Handbook Avidin and streptavidin conjugates—Section 7.6 antibodies, avidins and streptavidins, fluorescence, labeling and detection, secondary antibodies, secondary detection, signal amplification
Pierce Protein Methods Overview of protein labeling antibodies, antibody labeling, cell signaling, fluorescent dyes, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), protein labeling
Scientific poster (2007) Evaluation of click chemistry-based alternative to BrdU antibody labeling in tissue and cultured cells using fluorescence microscopy and flow cytometry  antibodies, ArrayScan, cell cycle, cell proliferation, Click-iT, flow cytometer/flow cytometry, fluorescence microscopy/fluorescence imaging, fluorescent dyes, microplate reader, nucleic acid labeling
Scientific poster (2007) Click catalyzed nucleic acid labeling as a novel replacement for BrdU antibody-based cell proliferation assay  Alexa Fluor, antibodies, antibody labeling, cell cycle, cell proliferation, Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, high content analysis, nucleic acid labeling
Scientific poster (2008) Zenon labeling technology facilitates detection of intra-cellular phosphoprotein determinants  Alexa Fluor, antibodies, cell signaling, flow cytometer/flow cytometry, fluorescent dyes, protein labeling, rare cell detection, Zenon
Scientific poster (2008) Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry  Alexa Fluor, antibodies, ArrayScan, cell cycle, Click-iT, flow cytometer/flow cytometry, fluorescent dyes, high content analysis, microplate reader, nucleic acid labeling, nucleic acid quantitation
Scientific poster (2008) ThiolTracker Violet dye, a new 405 nm excitable fluorescent probe for detecting intracellular thiols in imaging-based assays  ArrayScan, fluorescence microscopy/fluorescence imaging, fluorescent dyes, gel electrophoresis, glutathione, high content analysis, microplate reader, protein labeling, violet laser-excited reagents
Scientific poster (2008) Modified tumor cell protein expression and modification under physiological oxygen  Alexa Fluor, cancer, Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, gel electrophoresis, physiological oxygen level, protein labeling
Scientific poster (2008) A new 405 nm excitable fluorescent probe of intracellular thiols for imaging and flow cytometric assays  ArrayScan, flow cytometer/flow cytometry, fluorescence microscopy/fluorescence imaging, fluorescent dyes, glutathione, high content analysis, microplate reader, thiol modification, violet laser-excited reagents
Scientific poster (2008) Targeting serum glycoprotein cancer biomarkers with click chemistry  cancer, Click-iT, Electrophoresis gel, fluorescent dyes, gel electrophoresis, protein labeling
Scientific poster (2008) Detection of DNA synthesis by automated microscopy and image analysis: Comparison of BRDU method and a new click chemistry-based EDU method  Alexa Fluor, antibodies, ArrayScan, cell cycle, cell proliferation, Click-iT, flow cytometer/flow cytometry, fluorescent dyes, high content analysis, microplate reader, nucleic acid labeling
Scientific poster (2009) Monitoring mitotic cells and DNA content by automated imaging and analysis  Alexa Fluor, cell cycle, Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, high content analysis, nucleic acid labeling, nucleic acid quantitation, viability
Scientific poster (2009) Click chemistry-based detection of nascent RNA synthesis using high content imaging and fluorescence microscopy  Alexa Fluor, ArrayScan, Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, high content analysis, microplate reader, nucleic acid labeling, viability
Scientific poster (2009) Non-radioactive targeting of multiple classes of protein post-translational modifications (PTMs) with click chemistry  cell signaling, Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, gel electrophoresis, protein detection, protein enrichment, protein labeling, western blotting
Scientific poster (2009) Quantitative analysis of genotoxicity and cytotoxicity to DNA damaging agents using high-content imaging  Alexa Fluor, antibodies, Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, high content analysis, nucleic acid labeling, nucleic acid quantitation, viability
Scientific poster (2009) Metabolic labeling and click chemistry detection of stem cell markers  Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, gel electrophoresis, protein labeling, stem cell research
Scientific poster (2010) Novel tools to enable high resolution gene expression by investigating the nascent transcriptome  Alexa Fluor, Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, gene expression, high content analysis, magnetic beads, nucleic acid labeling, PCR/qPCR, viability
Scientific poster (2010) Click chemistry-based enrichment and identification of nascent and post-translationally modified proteins  cells, Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, gel electrophoresis, protein detection, protein enrichment, protein labeling
Scientific poster (2010) Non-cytotoxic near-IR DNA stain for cell cycle analysis in living cells  cell cycle, flow cytometer/flow cytometry, fluorescence microscopy/fluorescence imaging, fluorescent dyes, fluorescent proteins, live-cell imaging, nucleic acid labeling, viability
Scientific poster (2010) Click chemistry enrichment of cell surface glycoproteins  cells, Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, gel electrophoresis, protein detection, protein enrichment, protein labeling
Scientific poster (2010) Identification and characterization of O-GlcNAc modification of galectin-1 in mesenchymal stem cells using click chemistry  Alexa Fluor, BacMam technology, Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, gel electrophoresis, immunocytochemistry (ICC), protein enrichment, protein labeling, western blotting
Scientific poster (2010) Click chemistry-based universal enrichment columns for global identification of azide-modified proteins  Alexa Fluor, Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, gel electrophoresis, protein detection, protein enrichment, protein labeling
Scientific poster (2010) A universal click chemistry-based enrichment column for the identification of multiple subclasses of PTM-modified proteins and newly synthesized proteins  Alexa Fluor, Click-iT, flow cytometer/flow cytometry, fluorescence microscopy/fluorescence imaging, fluorescent dyes, gel electrophoresis, protein detection, protein enrichment, protein labeling
Scientific poster (2011) Site-specific click chemistry-mediated labeling of antibody glycans using metabolic and enzymatic approaches  Alexa Fluor, antibodies, antibody labeling, Click-iT, fluorescent dyes, western blotting
Tutorial 3.2 Secondary antibody optimization–Fixed cell imaging: 5 steps for publication-quality images 
Secondary antibody detection protocols also need to be optimized for each primary antibody used.
antibody labeling, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), secondary antibodies, secondary antibody protocol, secondary detection
Tutorial 2.2 Primary antibody protocol optimization–Fixed cell imaging: 5 steps for publication-quality images 
Every primary antibody must be optimized separately. There are many protocols available, and it is important to understand a "one size fits all" approach gives inferior results, as every antibody is slightly different. Learn how to approach optimization.
antibody labeling, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), primary antibodies, primary antibody protocol
Tutorial 3.5 Dye choice and special concerns–Fixed cell imaging: 5 steps for publication-quality images 
There are many different dyes spanning the visible, far-red, and infrared wavelengths.Considerations for making the right choices for your experiment are presented.
dye choice, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, Fluorescence SpectraViewer, immunofluorescence (IF), label choice, muliparametric dye selection
Tutorial 2.1 Primary antibody choice–Fixed cell imaging: 5 steps for publication-quality images 
After preparation, the second step to publishable images is to label the sample, usually involving primary antibodies to your specific targets of interest. The antibody source, the use of direct versus labeled antibodies as well as the validation for specific applications is discussed.
antibody labeling, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), primary antibodies
Tutorial 3.1 Secondary antibody choice–Fixed cell imaging: 5 steps for publication-quality images 
Step three of the five steps in making publishable images is to detect the label. That is, to detect with a secondary antibody, for instance, or an amplification technique, as well as to determine what controls to use. Your options are discussed.
antibody labeling, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), secondary antibodies, secondary antibody, secondary detection
Tutorial Labeling a purified antibody  Educational video on how to label your next antibody for imaging or fluorescence. 
Walk through the tutorial as Molecular Probes scientists demonstrate the protocol—including all the tips and tricks you'll want to know about for your next antibody labeling experiment. The video features Judie showing Curtis, a chemistry graduate student, how to label his monoclonal antibodies while saving time and maximizing yield.
antibodies, antibody labeling, fluorescence microscopy/fluorescence imaging, fluorescent dyes, immunofluorescence (IF)
Video How to site specifically label your antibody using SiteClick technology 
This video explains how to easily and site specifically label an antibody using an enzymatic and Click chemistry approach. This method can be applied to any intact IgG antibody and requires no antibody engineering or complex methodology. Unlike classic antibody conjugation techniques this breakthrough SitClick technology allows for antibody conjugation with complete confidence that the label will not directly interfere with the antibody binding domain.
antibodies, antibody labeling, flow cytometer/flow cytometry, fluorescence microscopy/fluorescence imaging, fluorescent dyes, Qdot, western detection
Webinar Fixed cell imaging—Five steps for publication-quality images 
With over 40 years dedicated to cell imaging research, we offer long-proven tools and protocols to help confidently create quality cell images the first time. This on demand webinar covers the 5 essential steps to getting great images.
antibodies, blocking, Celleste, dyes, EVOS FL Auto 2.0, fixation, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), sample detection, sample labeling, sample preparation, signal amplification
Webinar Learn to choose the right fluorophore when designing experiments 
The choice of fluorophore is one of the first important decisions to make in developing an experiment. Fluorophores are compounds that emit light at a specific wavelength when they have been excited at another, lower wavelength. Join our webinar and explore: How to choose the best organic dye for an assay Quantum dots and how they compare to other dyes When to use a phycobiliprotein like R-PE or APC When to use fluorescent proteins like GFP How to choose a suitable dye to match your instrument In addition, we will explore the basic characteristics, strengths, and weaknesses of the various fluorophores to help you choose and develop the best assay for your needs.
antibodies, antibody labeling, flow cytometer/flow cytometry, fluorescence microscopy/fluorescence imaging, fluorescent dyes, fluorescent proteins
Webinar A practical approach to antibody labeling 
The growing number of fluorophores available makes labeling your own antibodies a tempting proposition. But with many antibody labeling solutions available, selecting the best option can be a daunting task. In this webinar we will: Provide an overview of our antibody labeling kits Offer guidance on which methods are ideal for specific applications and experiments Provide tips and tricks to optimize your labeling protocol
antibodies, antibody labeling, flow cytometer/flow cytometry, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF)
Webinar An introduction to immunofluorescence staining of cultured cells 
In this webinar, we discuss the steps of an immunofluorescent staining protocol including material list, common variations, and necessary controls. We'll also provide a simple troubleshooting guide and examine how to avoid common pitfalls. Presented by Jason Kilgore, Technical Support Specialist, Thermo Fisher Scientific.
antibodies, antibody labeling,antifades, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF)
Webinar A comparison of basic immunofluorescent labeling strategies 
In this free webinar, we will compare different immunofluorescent labeling strategies exploring the pros and cons of each method. You will learn when the use of a direct conjugate is appropriate and when amplification techniques can be utilized. We'll also present a simple decision tree to aid in determining the best method for each situation.
Alexa Fluor, antibodies, antibody labeling, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunocytochemistry (ICC), immunofluorescence (IF)