western blot question

[AK]

Becky, are you sure your protein of interest is present in the cells you are 
using? your cell lysate is your best control. if the protein is not 
detectable in the cell lysate by Western, probably its not there. and if you 
expect to see it on gel, I suppose a lot of it should be there. Secondly, 
you are not sure if your antibody can detect it in Western, remember, 
sometimes antibodies which worked in one procedure may not work in other, 
check to see if it was tested in the specie you are using, also sometimes 
antibodies don't like hard boiled lysate or IP sample, also sometimes 
blocking with milk works sometimes BSA. To test antibody, purified protein 
would be the best control, but any known source would do. hth
AK
"Rebecca Pickin" <RPickin from cvm.msstate.edu> wrote in message 
news:mailman.255.1184203127.11350.methods from net.bio.net...
> 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
>
>