mpc at mrc-lmb.cam.ac.uk
Wed Nov 18 11:59:33 EST 1998
> Dear all
> I am trying to separate 600-700kD protein complexes from brain homogenate
> on a non-denaturing gel. However it has not been successful. I reallyneeds
> some suggestions from you.
> I use 4-12% gel including 0.1% chaps instead of SDS. The running buffer is
> Tris, glycine, and 0.1% chaps, and it is circulated during running PAGE.
> The samples (25-100 micro g on each lane of a regular size gel) are run
> 24-48 hrs at constant volt at 40, or constant current at 20mA, then
> stained by Coomassie blue. Every solubulized samples (by SDS, RIPA, chaps)
> were stuck on top of the stacking gel, did not get into the gel. However
> the marker went through. I do not know what do to next.
> If you have some suggestions, I really appreciate.
> Please respond e-mail to yumiko at mail utexas.edu
In removing the SDS you have removed the negative charge from the
proteins which causes them to migrate towards the anode. I've never
used CHAPS so I'm not sure whether it is ionic and performs the same
function, or, if it is, whether it is as effective as SDS? If your
protein complexes are disulphide-bonded (rather than 'domain
oligomerized') you could simply run the SDS-PAGE according to the
standard protocol, just omitting the beta-mercaptoethanol or DTT.
Hope this helps,
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