Biotinylation Protocol for Proteins or Peptides

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Biotinylation Protocol for Proteins or Peptides

1. If using biotin-NHS to conjugate to proteins, do not use Tris as the buffer, as it contains Nitrogen groups and can quench the reaction. PBS or HEPES as buffers are fine.
2. Dissolve the biotin in DMSO at 1 mg/ml just before use.
3. Add the the protein or peptide solution at a 1/10 dilution, and incubate on ice for 30 minutes or room temperature for 2 hours at a pH of 7.5-8.5 for biotin-NHS and at a pH of 6.5-7.5 for biotin-maleimide or biotin-BMCC.
4. Quench the reaction with Tris for biotin-NHS, and with beta-mercaptoethanol or dithiothreitol for biotin-maleimide or biotin-BMCC.
5. The biotin-tagged protein or peptide can be purified on a streptavidin column, or run on a protein gel and blotted with streptavidin-conjugated enzymes.