Hi All,
I am currently purifying a in vivo biotinylated protein from E. coli. It has a C-terminal Avitag (to allow in vivo biotinylation). I found that when I switch the buffer to lower salt (500-->150) and lower PH (8-->7), it start to precipitate out from the solution.
I have purified the non-biotinylated version of this protein and it behaved nicely and is very stable. Therefore, I think it was the added biotin that make it unstable.
Anyone has similar problem before? What is the best buffer to store biotinylated proteins?
Thanks ahead for your suggestions.
two things you may try, different salt concentrations and pHs. Many biological analysis can tolerant up to 500 mM NaCl, 300 mM is common to use as well. And keep buffer pH away from your protein pI.
Hello
Try to exchange the buffer stepwise. In a firt step change the pH of the bufffer by gel filtration or dialysis and then lower the salt concentration by slow dialysis. It should be efficient !
Good luck
Hi Ziguo Zhang,
Thanks a lot for your suggestion! My protein has a PI of ~7.66 and I am storing it at pH = 7. Do you think its far enough away from its PI? Its true that I can try storing it at higher salt while dilute the protein to lower salt for the assay. Good point.
Hi Claude,
I will try to dialyze my protein stepwise next time.. Maybe just too much changes in too short time for my protein to survive. Thank you!
NOTE:
I found that even though in this current buffer (50mM HEPES, 150 mM NaCl) some protein precipitate out from solution after dialysis, after I spinned down the aggregates there was still quite a bit of protein left in the supernatant. I'll try to use for my assay (AlphaScreen) tomorrow and hopefully it will work normally..
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