western blot question

Rebecca Pickin via methods%40net.bio.net (by RPickin from cvm.msstate.edu)
Wed Jul 11 09:05:12 EST 2007


Hello all!

I am trying to get an immunoprecipitation protocol up and running and am
in need of some help.  I have plenty of IP protocols, but I'm running my
results on a gel before I proceed with the next step.  Eventually, we
would like to be able to skip the gel.  But as of now, there is nothing
detectable on the gel, so we dont' want to proceed until we determine what
is wrong.  

Here's what is up so far:  I am running duplicate samples on both native
and SDS-PAGE gels.  When we silver stain the native gel, there are smears
and bands visible.  However, the only bands visible on the SDS-PAGE
western blot (detected with my antibody) are the markers.  

Samples include the initial cell pellet and lysate (which should contain
my protein of interest), pre-clearing and post-pre-clearing samples (which
may contain my protein), several washes (which shouldn't contain my
protein) and finally the beads (which should have my protein attached).  I
am concerned that my sample buffer for the native gel is not strong enough
to detach my protein from the beads.  Otherwise, the native looks like it
is working.

However the western blot with the SDS-PAGE gel has something wrong.  The
gel and buffers are fresh.  The markers transferred evenly and beautifly,
so I am thinking the transfer worked.  There could be something in the
detection step...  I guess my big question is..what could I run on the gel
as a control or to check my antibodies (although they are listed to be
good for Flow, WB, IP, and something else;  and the 1o worked for Flow and
is the one I'm using in the IP).  

Thanks for the help,
Becky




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