Site-specific biotinylation, typically via an AviTag and BirA ligase, attaches a single biotin at a defined position. This keeps the protein's binding surface unobstructed. Random amine labelling with NHS-biotin reagents modifies multiple lysines unpredictably. This can reduce activity if a lysine sits near the active site.
Proteins
Protein Labelling Kits for Site-Specific Biotinylation
Protein labelling kits for site-specific biotinylation and streptavidin-based detection
Enzymatic biotinylation attaches a single biotin tag to antibodies or recombinant proteins at a defined site, without disrupting antigen-binding or catalytic activity. This precision suits your pull-down assays, SPR, and other streptavidin-based workflows.
Enzymatic biotinylation uses E. coli biotin ligase (BirA) to attach a single biotin molecule to a 15-amino-acid AviTag sequence. The reaction occurs at a defined lysine within the tag, so every molecule you label carries exactly one biotin. This gives you consistent streptavidin-binding stoichiometry across experiments, which matters in SPR and single-molecule assays, where multi-site biotinylation creates orientation artefacts.
In vitro kits biotinylate your purified AviTag-fusion proteins directly. Companion E. coli expression strains support in vivo biotinylation during recombinant expression, removing the post-purification enzyme incubation step from your high-throughput protein production.
Biotinylation format depends on the workflow stage.
1) In vitro biotinylation of purified protein. A BirA Biotin Protein Ligase Kit suits your protein once it is purified and carries an AviTag. The kit adds BirA enzyme and biotin ATP cofactor, a 30-minute incubation at 30°C, then gel filtration removes the enzyme.
2) In vivo biotinylation during E. coli expression uses an E. coli expression strain that co-expresses BirA. The strain background matches your expression vector: XL1-Blue-background strains suit standard cloning vectors, BL21-type-background strains suit high-expression vectors, and K12-background strains suit K12-derived vectors. A 50 µM biotin supplement in the culture medium drives complete in vivo biotinylation.
3) Random amine labelling with NHS-biotin is not supplied by BioCat. When site-specific labelling is not required and your protein has no AviTag, commercial NHS-biotin reagents from standard biochemical suppliers fit this route.
Applications
Immunoassay and detection
Site-specifically biotinylated proteins on streptavidin surfaces give you consistent orientation for sandwich ELISA kits, lateral flow, and biosensor assays.
Affinity pull-down and proximity labelling
Biotinylated bait proteins immobilised on streptavidin beads let you identify interacting partners by co-precipitation followed by mass spectrometry.
Cell-surface receptor tracking
Labelled extracellular proteins or antibody fragments let you track receptor internalisation and recycling using live-cell imaging tools.
SPR and BLI binding kinetics
Uniform site-specific biotinylation via AviTag gives you consistent capture density on SPR biosensor chips, for reliable on-rate and off-rate measurements.
Single-molecule and super-resolution studies
One biotin per molecule gives predictable, low-density immobilisation on streptavidin surfaces, avoiding multi-site artefacts.
High-throughput recombinant protein production
In vivo biotinylation during E. coli expression skips the post-purification labelling step, streamlining your AviTag-fusion protein panels.
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Protein Labelling
Site-Specific Biotinylation Kits for Consistent Streptavidin Detection
Frequently asked questions
Yes. NHS-biotin reagents react with primary amines on lysine residues and the protein N-terminus, without requiring any tag. The trade-off is that labelling is random and the degree of substitution varies between batches. For assays sensitive to protein orientation, adding an AviTag during construct design gives better reproducibility.
Run a streptavidin-HRP dot blot on the labelled protein alongside an unlabelled control. A band or dot visible only in the labelled lane confirms biotin incorporation. For quantitative applications, an HABA displacement assay or a biotin-quantification kit gives the average moles of biotin per mole of protein.
Completeness depends on biotin availability in the culture medium. Supplementing with 50 µM biotin and inducing at low temperature (16 to 20°C) improves stoichiometry. A streptavidin gel-shift assay, where fully biotinylated protein runs slower after streptavidin addition, confirms the fraction that is labelled.
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