SDS-PAGE-resistant protein complexes
Elmer M. Price, Ph.D.
DCRCEP at mizzou1.missouri.edu
Tue Jun 21 15:20:40 EST 1994
In article <rosto001.772140525 at maroon>
rosto001 at maroon.tc.umn.edu (Alexander P Rostovtsev) writes:
>In <Mailstrom.1.03.12087.15089.agibson at acs.ucalgary.ca> agibson at ACS.UCALGARY.CA (Allan Gibson) writes:
>>Hi! Has anyone on the net ever had experience with protein complexes that are
>>not dissociated under normal SDS-PAGE conditions, i.e. brief boiling in SDS
>>sample buffer containing 2% SDS and 5% 2-mercaptoethanol. If so, I'd appreciate
>>very much hearing from you and finding what conditions, if any, might break such
>>a complex into its components. Thanks.
> Don't boil samples. Try 30-45 min incubation at 60-70oC or
>overnight at RT.
We use a slightly different protein sample buffer recipe which contains
6M urea, 2%SDS, 160mM DTT, 62.5mM Tris pH 6.8. Then, definitely do not
boil, or even heat to 70 degreec C. In our hands, our favorite protein
will aggregate if heated to just 37 degrees. An incubation at room temperature
(which is unfortunately about 85degreesF at this time) for at least 30 min
with occasional vortexing works well.
Hope this helps
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