SDS-PAGE:slower migration after reducing?

Ron Tate rtate at bmb-fs1.biochem.okstate.edu
Thu Jan 25 14:22:10 EST 1996


John Edward Smith wrote:
> 
> Any ideas why the same sample treated with the same loading buffer (2X conc:
>  0.125 M Tris pH 6.8, 4% SDS, 20% glycerol) either (1) without beta-
> mercaptoethanol and without boiling or (2) with BME (5% final conc) and
>  boiling 10 minutes results in a slower migration under reducing/denatureing
> conditions?
> 
> Many thanks
> -John Smith

Without the BME and boiling you've most likely got S-S in the polypep so 
a more compact molecule (not fully extended) which is not fully 
accessable to SDS and thus less negative charge per the same molecular 
weight and thus slower migration.  Also without adequate reducing power 
of the sample you are more likely to see extra bands from differant 
degrees of disulfide reduction of the same proteins.

Happy Hunting
Ron Tate
OSU BMB



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