immunoprecipitation and 2D gels

Stephen Dahl stayve-and-irayne at worldnet.att.net
Fri Oct 27 21:29:30 EST 2000



Am I correct when I read the last sentence of your post that when you
resuspend the beads in a small amount of your Urea/ampholytes plus SDS,
spin, remove the sup and run 1-D SDS-PAGE that you can see the sample, but
if you take an aliquot of the same sample on 2-D you see nothing??   Have
you tried staining the IEF tube gel to see if the protein is entering the
gel bed?  You say this used to work, but now doesn't which in my mind
implicates either the IEF gel (acrylamide conc. mistakenly too high?) or the
ampholytes have gone funky (for lack of a better word) and the sample is
refusing to enter the gel.  If I have mis-read your last sentence, then I
would first check and make sure the sample is leaving the beads in the
Urea/ampholyte solution.

Good luck,
Steve Dahl

Alistair J Lax <alistair.lax at kcl.ac.uk> wrote in message
news:SIMEON.10010271646.F at oralp-13.umds.ac.uk...
> We are having problems visualising protein spots on 2D gels
> after immunoprecipitation, and would appreciate any
> suggestions. The protein is immunoprecipitated, and the
> beads then added to the first dimension buffer (urea and
> ampholites) and loaded onto the Biorad IEF cell. After the
> second dimension, the SDS PAGE gel is silver stained. This
> worked well for a couple of months, but all we get now are
> some lower molecular weight contaminants. Beads loaded
> straight onto SDS PAGE show the protein - either in blots
> or with silver stain. The early stages of the prep have
> protease inhibitors added, and we made all the solutions
> again after this problem appeared. We have tried adding SDS
> to the first dimension elution buffer without success. The
> protein is there when we elute the protein from the beads
> in SDS followed by removal of beads, that is it can be seen
> by SDS PAGE, but not in 2D gels.
>
> Alistair Lax
>
> alistair.lax at kcl.ac.uk
> King's College London
>
>
> ---







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