Overview of Protein Labeling

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Biotin is a useful label for protein detection, purification and immobilization because of its extraordinarily strong binding to avidin, streptavidin or Thermo Scientific NeutrAvidin Protein. Indeed, this interaction is one of the strongest noncovalent interactions between a protein and ligand. Additionally, biotin (244.3 Da) is considerably smaller than enzyme labels and is therefore less likely to interfere with normal protein function. Together, these features make avidin–biotin strategies ideal for many detection and immobilization applications. However, depending on the nature of the application, the very strong binding interaction can be problematic. In those situations, certain variants of avidin or derivatives of biotin are available, which allow soft-release (elution) binding or cleavable (reversible) labeling.

Biotinylation is the process of labeling proteins or nucleotides with biotin molecules and can be performed by enzymatic and chemical means. Chemical methods of biotinylation are most commonly used, and the biotinylation reagents used for this type of labeling share several basic features. They are composed of the biotinyl group, a spacer arm and a reactive group that is responsible for attachment to target functional groups on proteins. Variations in these three features account for the many varieties of available reagents and provide the specific properties needed for particular applications.

Spacer arms link the biotin molecule to a reactive group that interacts with certain functional groups on the amino acids of the target protein. Besides connecting biotin to a chemical group that mediates protein attachment, spacer arms can influence biotinylation and protein detection in three ways. First as indicated in the following diagram, these spacer arms vary by length, which can affect the availability of the attached biotin for binding to avidin, streptavidin or NeutrAvidin.

Examples of variable spacer arm lengths. Chemical groups (black) modify the distance between the reactive moiety (red) and biotin (blue) to regulate the length of the spacer arm. The reagents shown are (A) Thermo Scientific EZ-Link NHS-Biotin, (B) EZ-Link NHS-LC-Biotin and (C) EZ-Link Sulfo-NHS-LC-LC-Biotin.

Second, the solubility of a biotinylation reagent is an important factor that can influence its ability to biotinylate proteins that are located in membrane-bound compartments or alter the solubility of the labeled target protein. For example, a spacer arm consisting of poly(ethylene) glycol (PEG) repeats will increase or preserve the solubility of labeled proteins.

Poly(ethylene glycol) increases the solubility of biotinylation reagents. A four-ethylene glycol chain (PEG4) was conjugated onto the spacer arm between the reactive moiety (red) and biotin (blue). The reagent shown is Thermo Scientific EZ-Link NHS-PEG4-Biotin.

In contrast, long hydrophobic spacer arms can render a labeled target protein less soluble but are ideal when performing labeling reaction in hydrophobic organic solvents such as dimethylsulfoxide (DMSO), which is often required when making modifying hydrophobic peptides. Third, spacer arms may contain a cleavable region (e.g., a reducible disulfide bond) that mediates separation of the biotin label from the protein to allow purification without harsh denaturants.

A wide range of biotinylation reagents with different reactive groups are commercially available. Common reactive groups and their respective targets on proteins include:

• N-hydroxysuccinimide (NHS) and Sulfo-NHS—primary amines
• Maleimide, iodoacetyl groups or pyridyl disulfides—sulfhydryls
• Primary amines in combination with EDC—carboxyls
• Hydrazines and alkoxyamines—glycoproteins

Additionally, photoactivatable aryl azides can be used to mediate non-selective biotinylation upon exposure to UV light.

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Active site probes are a class of chemical labeling reagents whose reactive groups are designed to specifically bind (label) particular enzyme active sites. Similar to traditional chemical labeling probes, active site probes contain a detectable tag (biotin/dye), a spacer arm and a reactive group that is responsible for attachment to the active site of the target class of enzymes. Active site reactive groups are typically electrophilic compounds which covalently link to nucleophilic residues found in enzyme active sites. In cases where the active site reactive group does not covalently bond to the target enzyme, photoreactive groups are incorporated into the linker region to facilitate attachment following specific binding. These probes can be used to selectively enrich, identify and profile target enzyme classes across samples or assess the specificity and affinity of enzyme inhibitors.

Active site probes have been developed to label different specific enzyme classes such as kinases, phosphatases, GTPases, serine hydrolases, cysteine proteases, metalloproteases and cytochrome p450 enzymes. All active site probes can be used to determine inhibition of enzymes by small molecules, and some probes also preferentially react with only active enzymes, allowing for activity-based proteomic profiling (ABPP). ABPP is a powerful method to monitor protein activity versus traditional protein or RNA expression profiling techniques that only measure abundance. The follow image and table illustrates processes involved in the detection of active serine hydrolase enzymes, and provides a list of serine hydrolases identified by mass spectrometry, respectively.

Mechanism and chemical structures of active site probes for serine hydrolases. (A) Fluorophosphonate probes (FP) covalently and specifically attach to the active site serine of active serine hydrolases and proteases. (B) Structures of the azido, desthiobiotin and fluorescently-tagged fluorophosphonate probes for labeling, affinity enrichment or fluorescence detection of active serine hydrolase enzymes.

Serine hydrolases identified by mass spectrometry with ActivX fluorophosphonate (FP) probes. Number of serine hydrolase family members from mouse brain and liver tissue extracts identified by mass spectrometry after labeling and enrichment using the desthiobiotin-FP probe.

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Certain enzymes have properties that enable them to function as highly sensitive probes with a long shelf life and versatility for the detection of proteins in tissues, whole cells or lysates. Enzyme labels are considerably larger than biotin and require the addition of a substrate to generate a chromogenic, chemiluminescent or fluorescent signal that can be detected by different approaches. Enzyme labels are widely used because of their multiple types of signal output, signal amplification and the wide selection of enzyme-labeled products, especially antibodies.

Enzymes commonly used as labels include horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase and β-galactosidase, and specific substrates are available for each enzyme. Indeed, multiple commercial substrates are available for HRP and AP that generate colorimetric, chemiluminescent or fluorescent signal outputs. To generate the following data, an anti-mouse IgG-HRP conjugated secondary antibody was used to perform immunohistochemistry (IHC).

Detection of p21 in human lung colon carcinoma by IHC. IHC staining for p21 in a formalin-fixed paraffin-embedded (FFPE) section of human colon carcinoma using a monoclonal antibody as the primary antibody and an anti-mouse IgG-HRP conjugate as the secondary antibody. The brown precipitating HRP substrate DAB was used. Prior to staining, heat-induced epitope retrieval (HIER) was performed in 10 mM citrate buffer.

Enzyme probes can be conjugated to antibodies, streptavidin or other target proteins by multiple mechanisms, including glutaraldehyde, reductive amination following periodate oxidation of sugars to reactive aldehydes, or by using heterobifunctional crosslinkers such as Sulfo-SMCC.

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Fluorescent reagents of many types continue to be developed to stain or chemically label proteins, nucleic acids and other biomolecules. When specific antibodies or other purified biomolecules are chemically labeled with fluorescent dyes, they become fluorescent probes for detection of target antigens or interaction partners in applications such as cell imaging, high-content analysis, flow cytometry, western blotting and ELISA.

The following representative examples include immunohistochemistry (IHC) and immunocytochemistry (ICC) data generated using multiple fluorescently-labeled probes that allow researchers to identify a variety of structures within the tissue or cell, respectively.

Immunocytochemistry analysis of adiponectin in HeLa cells. This experiment was performed using an Invitrogen ABfinity adiponectin recombinant rabbit monoclonal antibody followed by detection using an Invitrogen Alexa Fluor 488–conjugated goat anti-rabbit secondary antibody (green) (A). Nuclei were stained using DAPI (B) and actin stained with Alexa Fluor 594 phalloidin (red) (C). Image D is a composite image showing subcellular localization in the perinuclear region.

Detection of cytokeratin 18 in human colon carcinoma tissue by IHC using immunofluorescence. The sections were incubated with a biotinylated anti–cytokeratin 18 antibody and then detected using an Invitrogen streptavidin–DyLight 633 conjugate (red fluorescence). Invitrogen Hoechst stain was used to counterstain the cell nuclei (blue fluorescence).

Fluorescent molecules, also called fluorophores or simply fluors, respond directly and distinctly to light and produce a detectable signal. Unlike enzymes or biotin, fluorescent labels do not require additional reagents for detection. This feature makes fluorophores extremely versatile and the new standard in detecting protein location and activation, identifying protein complex formation and conformational changes, and monitoring biological processes in vivo.

The current vast selection of fluorophores provides greater flexibility, variation and performance for research applications than ever before. Fluorophores can be divided into three general groups, and each group of probes has distinct characteristics. These groups are as follows:

• Organic dyes—Alexa dyes, FITC, TRITC, DyLight fluors
• Biological fluorophores—Green fluorescent protein (GFP), R-phycoerythrin
• Quantum dots

Our broad portfolio of fluorescent reagents includes Invitrogen Alexa Fluor dyes, as well as various traditional and specialty fluors associated with our Invitrogen Molecular Probes and Thermo Scientific Pierce product lines.

Detection of fluorescent probes requires specialized equipment, including an excitation light source, filter set and a detector, which are found in fluorescence microscopes, fluorescence plate-readers, flow cytometers and cell sorters. This equipment enables the absolute quantitation of proteins based on fluorescence, which is a significant benefit to using fluorescent probes over other types of probes.

Jablonski energy diagram of fluorescence. When a fluorophore is excited by an appropriate wavelength of light, it will absorb the light, resulting in a loss of some of the absorbed energy, along with emission of light at a higher wavelength, and finally a return to the ground state of the molecule. 

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Both in vitro and in vivo methods of protein and nucleic acid labeling have been developed to accommodate the need for all types of biomolecular probes.

Chemical methods of protein labeling involve the covalent attachment of the label to amino acids using a label conjugated to chemical groups that react with specific amino acids. These reactive groups, described in detail in the Crosslinker section of the Pierce Protein Methods library, react with specific moieties on distinct amino acids, although a few are also available that nonspecifically react with any amino acid at C-H and N-H bonds. These reactive groups are also used to label nucleic acids.

Enzymatic methods are also used to label both proteins and nucleic acids. These in vitro methods require the respective polymerases, ATP and labeled amino acids or nucleotides. While in vitro DNA transcription is relatively straightforward, the expression of labeled proteins by in vitro translation can be difficult because of the requirement for proper protein length, folding and post-translational modifications that some commercial kits are unable to provide.

Enzymatic labeling of RNA at the 3’ hydroxyl end using a biotinylated cytidine biphosphate (pCp-biotin) via the use of T4 RNA ligase and ATP.

Metabolic labeling is a method to label all nucleic acids or proteins in a cell by culturing them with labeled nucleotides or amino acids, respectively. Prolonged cell culture in media containing labeled nucleic acids or amino acids results in all DNA, RNA or proteins becoming labeled via DNA replication, translation, and protein turnover. The nucleic acid or protein of interest can then be purified for further experimentation. The benefit of performing metabolic labeling is the consistent labeling of all nucleic acid or protein species. Conversely, metabolic labeling can be toxic, depending on the type of label used, and the number of metabolic labeling reagents is not as broad as those for in vitro methods.

Comparison of several in vivo crosslinking methods. HeLa cells were treated with 1% formaldehyde (HCHO) or 1 mM homobifunctional NHS-ester crosslinker (Thermo Scientific Pierce DSG and DSS) in PBS for 10 minutes before quenching. A fourth set of HeLa cells were treated and crosslinked for 10 minutes with 4 mM Photo-Leucine, 2 mM Photo-Methionine (Photo-AA) according to the procedure. Formaldehyde-treated and NHS-ester–treated cells were quenched with 100 mM glycine (pH 3) and 500 mM Tris (pH 8.0), respectively for an additional 15 minutes. One million cells from each condition were then lysed and 10 µg of each sample was heated at 65°C for 10 minutes in reducing sample buffer containing 50 mM DTT, followed by analysis by SDS-PAGE and western blotting with Stat3-specific antibodies (Cell Signaling). Gapdh (Santa Cruz) and beta-actin (US Biologicals) were blotted as loading controls.

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1. Green NM (1963) Avidin. 3. The nature of the biotin-binding site. Biochem J (9):599–609.
2. Green NM, Toms EJ (1973) The properties of subunits of avidin coupled to Sepharose. Biochem J (133):687–698.
3. IUPAC Commission on Photochemistry (1996) Pure Appl Chem 6(8):2223–2286.