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Overview of Protein Labeling

Biological research often requires the use of molecular labels that are covalently attached to a protein of interest to facilitate detection or purification of the labeled protein and/or its binding partners. 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. Therefore, the type of label and the labeling strategy used must be carefully considered and tailored for each application.

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Protein Methods Library Home

Protein Labels

Biotin

Biotin is a useful label for protein detection, purification and immobilization because of its extraordinarily strong binding to avidin, streptavidin or NeutrAvidin Protein. Indeed, this interaction is one of the strongest non-covalent 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, these spacer arms vary by length, which can affect the availability of the attached biotin for binding to avidin, streptavidin or NeutrAvidin. Second, the solubility of a biotinylation reagent is an important factor that can influence the 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. 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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Biotinylation (includes disussion of cleavable and reversible biotin)

Overview of the Avidin-Biotin Interaction

 

Related literature...

Avidin-Biotin Handbook

Active Site Probes (enzymes as the target of labeling)

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, photo-reactive 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). Activity-based proteomic profiling is a powerful method to monitor protein activity versus traditional protein or RNA expression profiling techniques which only measure abundance.

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Enzyme Active Site Probes

Enzyme Conjugates (enzyme reporters as the detection tag)

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.

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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Enzyme Probes

Fluorescent Probes

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 vast selection of fluorophores today provides greater flexibility, variation and fluorophore 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 – FITC, TRITC, DyLight Fluors
  • Biological fluorophores – Green fluorescent protein (GFP), R-Phycoerythrin
  • Quantum dots

Detection of fluorescent probes requires specialized equipment, including an excitation light source, filter set and a detector, that 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.

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Fluorescent Probes

Protein Labeling Strategies

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.

 

In vitro Labeling

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 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.

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DNA and RNA Labeling

Overview of Crosslinking

In vivo Labeling

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.

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Cell-free in vitro Recombinant Protein Expression

Metabolic Labeling

Written and/or reviewed by Jared Snider, Ph.D.

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