Kit supplied as:
• Ten (10) vials of 10 µL each 3mg/mL BirA biotin-protein ligase (300 µg total)
• Ten (10) vials of 1.5 mL each BiomixA (0.5 M bicine buffer solution, pH 8.3)
• Ten (10) vials of 1.5 mL each BiomixB (100 mM ATP, 100 mM Mg2+acetate, 500 µM d-biotin)
• Ten (10) vials of 1.5 mL each additional BIO-200 (500 µM d-biotin)
The BirA biotin-protein ligase (EC 126.96.36.199) 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.
Does the BirA contain an affinity purification tags, such as the His-tag?
No. Our BirA is the wild-type purified via traditional protein purification methods from E. coli. BirA purified in this manner without fusion tags is the most active available.
What is the molecular weight of the BirA enzyme?
How much BirA do I need to biotinylate my AviTag’d protein?
The long answer to this question is that for every 10 nmol of substrate (at 40uM concentration in the final reaction mix) we recommend 2.5ug of BirA enzyme to complete the biotinylation in 30- 40 min. at 30°C. The short answer is we recommend about 1ug of BirA to every 100ug of AviTag’d peptide/protein for good rates of biotinylation. Enzyme dynamics are complicated in that lower biotinylation substrate concentrations (or lower BirA concentrations) increase reaction times significantly. For example, where 40uM of substrate may take 30 minutes to biotinylate to completion, 4uM substrate concentration may take 5 hours with the same amount of BirA. The best reaction conditions will be determined by the individual user.
Do I need to add additional d-biotin to the reaction mix?
Usually not. The BiomixB included in the BirA biotinylation reaction kit contains 500uM d-biotin for 50uM final concentration. The BIO200 (500uM d-biotin) also included in the BirA biotinylation reaction kit is for use with modified amounts of substrate in the biotinylation reaction. If you want to try to biotinylate larger amounts of substrate or greater than recommended concentrations, extra d-biotin (and extra BirA) can help decrease reaction times.
How long can I store the BirA enzyme and still maintain full functionality?
Our BirA enzyme is a very stable protein. It can be stored for years at -80 degrees Celsius and still have full functionality (only if continuously frozen!). Once thawed, unused portions can be flash-frozen in liquid nitrogen and stored again at -80 degrees Celsius with only a minimal loss of functionality (10-20% each time). If stored at 4 degrees Celsius after thawing, the BirA enzyme is functional for weeks. Count on perhaps a 5-10% loss in activity per week at 4 degrees Celsius. That being said, the most volatile part of the BirA biotinylation reaction kit is the BiomixB. BiomixB contains ATP which is subject to hydrolysis when not frozen. Generally when we are contacted about problems with the BirA reaction it is because the BiomixB has gone off. All is not lost however as additional ATP added to the reaction will start it back up again.
Is it necessary to quench the reaction or remove the BirA when the reaction is done?
No. The reaction is basically self-quenching. As the biotinylation nears completion it slows down as the BirA “searches” for unbiotinylated substrate. As long as there is ATP and d-biotin available and the ATP has not been exhausted, the reaction goes to completion. The BirA afterwards is inert. It will not react with anything else besides ATP and substrate. You can remove the BirA with size-exclusion chromatography or a cationic exchange resin (assuming your protein does not also stick to it) if you desire but it is not necessary.
How do you remove the excess free d-biotin?
We have found that using a simple desalting column is effective at removing the d-biotin since it is so small (244.3 Daltons).