Michael J. Tuvim
mtuvin at bcm.tmc.edu
Mon Mar 4 13:38:48 EST 1996
In article <Pine.3.89.9602240120.A17519-0100000 at mistral.ERE.UMontreal.CA> ferlandl at ERE.UMontreal.CA ("Ferland Louis H.") writes:
>Most people I know try to not load more than 50 micrograms. We tried
>loading 200 ug to detect a low abundance protein we couldn't purify and
>got only a mess. We finally had to find a different way to improve the
>limit of detection.
>On Fri, 23 Feb 1996, lmrc. wrote:
>> What is the maximum amount of total protein one can load on a PAGE gel
>> for Western analysis??
We routinely load 100-150 ug of total protein on our gels. If you make
your gels thicker (3 mm instead of 1,5) you don't have any smears and
smiles and generally end up with good enough resolution. The problem is
with the antibody: it should be VERY specific to ignore the plethora of
accompanying proteins. Secondary AB will also pick nonspecific bands, so
it might be worth trying several.
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