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FAQs
I am using the consensus peptide described by Schatz (1993)
but the levels of biotinylation are poor. How can I improve this?
The consensus sequence is, in effect, an average of the efficient and inefficient peptide sequences. The most efficient sequence is #85. (G L N D I F E A Q K I E W H E) You need to convert over to this.
Can the in vitro biotinylation be scaled up?
Yes. Milligram quantities of protein can be biotinylated in overnight reactions. A protocol is given in Crawford et al., Immunity 8, 675-682, 1998.
What percent of my protein can I expect to be biotinylated in vivo?
Using an E. coli strain that over-expresses birA such as AVB101, we see 85-95% biotinylation of substrate proteins.
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.
Do detergents inhibit birA?
We have tested Tween 20 at 0.1% concentration and not found any effect on the biotinylation reaction.
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.
How can I quantitate the level of biotinylation of my protein?
We have developed a simple 'dot blot' assay and plate assay .
I am preparing my own biotin solutions.
Why am I having problems with the biotinylation reaction?
We have noticed that the biotin from some suppliers tends to 'go off' rapidly. We routinely use biotin from Research Organics, as this is the most stable we have come across.
Can I achieve biotinylation of proteins bound to a nitrocellulose membrane?
This is something we have not tried. Please let us know if you have any experience with this kind of experimental set-up.
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