E. coli in vivo Biotinylation Kit (without BirA Competent Cells)
The E. coli in vivo Biotinylation Kit (without BirA Competent Cells) comes in a convenient, easy-to-use format and contains the components necessary to label biotin-acceptor peptides with biotin expressed in E. coli. strain BL21, a chemically competent E. coli B strain. This kit contains sufficient reagents to label 10 x 1 L of E. coli culture. BirA-Transformed chemically competent E. coli cells (BPS Bioscience #27462) are not provided with this kit and must be purchased separately.
- BirA-Transformed Chemically Competent coli (BPS Bioscience #27462)
- 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 |
| 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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