E. coli in vivo Biotinylation Kit (with BirA Competent Cells)
The E. coli in vivo Biotinylation Kit (with BirA Competent Cells) comes in a convenient, easy-to-use format and contains all the components necessary to label biotin-acceptor peptides with biotin in E. coli. strain BL21, a chemically competent E. coli B strain. The cells include an IPTG-inducible plasmid containing the BirA gene and a resistance gene for streptomycin or spectinomycin. This kit contains sufficient reagents, including BL21 cells, to express and label 10 x 1 L of E. coli culture.
Start by transforming the competent cells with a vector expressing your protein of interest linked to an AviTag™ sequence (for example: pAN or pAC vectors). After transformation, BirA expression is induced by IPTG and biotin is added to the culture. BirA efficiently catalyzes the specific attachment of biotin to the AviTag™.
- SOC or LB medium.
- Expression vector containing gene of interest and the AviTag™ sequence.
- Streptomycin or spectinomycin, and appropriate antibiotic based on expression vector for the gene of interest.
- Water bath
- Protease inhibitor cocktail (for example, protease cocktail from Sigma #P8465: AEBSF 23 mM, EDTA 100 mM, Bestatin 2 mM, Pepstatin A 0.3 mM, E-64 0.3 mM).
- Affinity chromatography for AviTag™
| Catalog # | Name | Amount | Storage |
| 27462 | BirA-transformed Chemically Competent E. coli |
10 x 50 µl | -80°C |
| 100x Biotin (5 mM) | 10 x 12 ml | -80°C | |
| 1000x IPTG (1M) | 10 x 1.3 ml | -80°C | |
| 1000x DTT (1M) | 10 x 150 µl | -80°C | |
| 10x Lysis Buffer | 10 x 6 ml | Room Temp |
Biotin-labeling is commonly used for non-radioactive labeling and purification of proteins or other target molecules. Biotin labeling takes advantage of the exceptionally strong interaction between biotin (vitamin H) with avidin or streptavidin. The affinity of biotin to avidin or streptavidin is one of the strongest known non-covalent interactions known between a protein and a ligand, exhibiting a dissociation constant (Kd) around 4x10-14 M. BirA, an E. coli biotin protein ligase, covalently adds biotin to an AviTag™ peptide in a highly efficient and specific manner, in a reaction that requires ATP. It has the advantage of resulting in a homogeneous product that can be used in protein capture, immobilization and functionalization.
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