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).
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 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.
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 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.
What is the molecular weight of the BirA enzyme?
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 are the best storage conditions for birA?
The birA enzyme arrives frozen. If you are not going to use it immediately, then store it at -80°C. Once the enzyme has been thawed, it should then be stored at 4°C. We do not recommend repeatedly re-freezing the enzyme as there is a 5-10% loss of activity during each freeze/thaw cycle. At 4°C the enzyme is stable for several months. For long-term storage, the enzyme can be safely re-frozen by dropping into liquid nitrogen before storing at -80°C.
Do detergents inhibit birA?
We have tested Tween 20 at 0.1% concentration and not found any effect on the biotinylation reaction.
Does your birA enzyme contain proteases?
The level of protease contamination of the birA enzyme is almost undetectable. If the target protein has not been extensively purified, then this is a much more likely source of proteases! If you are finding proteases to be a problem, we recommend adding a protease inhibitor cocktail. For proteins expressed in E. coli we recommend the Sigma protease inhibitor cocktail (catalog #P8849) at a 1/100 dilution. For mammalian or insect cell expression, we recommend a protease inhibitor cocktail containing leupeptin, pepstatin and PMSF to the biotinylation reaction mix.