protein reduction

Paul S. Brookes. brookes at uab.edu
Fri Jan 5 10:03:24 EST 2001


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

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