western of protein expressed from pGAD-424

Jackson, John C. JohnC.Jackson at anheuser-busch.com
Thu Sep 21 10:47:33 EST 2000


You might have to try a variety of lysis protocols to find the one which works
best.  Y-PER from Pierce Chemical is the easiest and in practice works very well
for soluble proteins.  If you're fusion protein is sequestered to some
structural element, it may not work so well.  In that case try the glass bead
method.  You also may have to do some ammonium sulfate cuts to concentrate your
extract, in which case I wouldn't recommend Y-PER because of salt issues.  As
far as the antibodies, I can't offer any help.  However, for detection of low
expressed proteins, try the Pierce SuperSignal West Femto reagent as it most
likely is the most sensitive chemiluminescent detection substrate out there.  Be
sure to follow Pierce recommendations on antibody dilution as well (not the
antibody vendors recs).
Hope this helps somewhat.

--John


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