We do a "careful" reduction of BSA (whatever that means). For a 50mg/ml
solution of BSA we use 100mM DTT, stirring on ice for 2hrs in the dark. I
think the length of time and the dark are quite important. Obviously
certain disulfides will only become accessible to the solvent/reductant
after other disulfides have been reduced, allowing the protein to
unfold. Bottom line, try leaving it for a bit longer.
This of course assumes that your eventual aim is to unfold the
protein. Another possibility is that it is energetically unfavorable for
your protein to unfold and break the final 1 or 2 disulfides, for example
due to hydrophobic interactions. Try adding a mild detergent such as
lauryl-maltoside.
Regards
PSB
_________________________________________
Dr. Paul S. Brookes. (brookes at uab.edu)
UAB Department of Pathology, G004 Volker Hall
1670 University Blvd., Birmingham AL 35294 USA
Tel (001) 205 934 1915 Fax (001) 205 934 1775
http://peir.path.uab.edu/brookes
The quality of e-mails can go down as well as up
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