Kit supplied with the following:
- Two (2) vials of 20uL each 1mg/mL BirA biotin-protein ligase (40ug total)
- Two (2) vials of 1.5mL each BiomixA (0.5M bicine buffer solution, pH 8.3)
- Two (2) vials of 1.5mL each BiomixB (100mM ATP, 100mM Mg(OAc)2, 500uM d-biotin)
- Two (2) vials of 1.5mL each additional BIO-200 (500uM d-biotin)
The BirA biotin-protein ligase (EC 220.127.116.11) adds d-biotin covalently to biotin-acceptor peptides/proteins via an ATP intermediate (biotinyl 5’-adenylate) in a highly efficient and targeted manner. The downstream applications of enzymatically biotinylated proteins are varied, important and powerful. The well known biotin-avidin/streptavidin interaction is often exploited for affinity chromatography or protein immobilization on surfaces or substrates. Protein detection via anti-biotin antibodies or avidin/streptavidin-reporter enzyme conjugates (-HRP, -alkaline phosphatase) or fluorescent probes becomes possible. Multimeric forms of biotinylated MHC molecules are popular tools in immunobiology.
When used in combination with our AviTagTM biotin-acceptor peptide amino acid sequence, biotinylation occurs at twice the rate of the natural E. coli BCCP substrate and as much as an order of magnitude or more over other Biotinylation of Peptide sequences. The AviTagTM sequence consequently requires less of the BirA enzyme and shorter incubation times to biotinylate to completion than do other sequences available. If protein instability or protease activities are a concern this may be important to downstream success.
Our BirA enzyme is E. coli wild-type, encoded by the birA gene, and purified to greater than 99% purity by traditional methods. Other names for this enzyme include: biotin ligase; biotin operon repressor protein; birA; biotin holoenzyme synthetase; biotin-[acetyl-CoA carboxylase] synthetase; biotin-[acetyl-CoA-carboxylase] ligase; biotin-[acetyl-CoA carboxylase] synthetase; acetyl CoA holocarboxylase synthetase; acetyl CoA holocarboxylase synthetase; biotin:apocarboxylase ligase; biotin holoenzyme synthetase; HCS.