how to concentrate Co-IP protein sample

tracy dreamwestus at yahoo.com
Tue Mar 16 13:06:43 EST 2004


Thank you very much for your reply.

My final goal is to identify some novel proteins that come down with this
HA-tagged protein using mass spectromety.
Do you know if excessive SDS will inhibit mass spec analysis?

I have tried eluting the protein with glycine (pH2.5), but at least on the
western, almost none of the protein got eluted.  I don't  why, I just simply
added
100ul of glycine to about 20ul of HA-beads, let it sit for about 5 minutes,
then spun down the beads and got the eluate.  Is it the right way to do it?


"P.C." <PC at nospam.edu> wrote in message
news:c35qmv$1if$1 at cruncher.dfci.harvard.edu...
> Microcon-3 (from Millipore) should be just fine (you will concentrate
> SDS too to some extent, but that does not matter too much).
> You could also elute the protein in acid (Gly-HCl, pH2), and then do TCA
> (may be first neutralize HCl would be safer (?)).
>
> On the other hand, it might be more efficient to upscale and use more
> concentrated extract in your IP with same amount of Ab. Did you check by
> western if you IP most of your HA-tagged protein?
>
> Peter
>
> tracy wrote:
> > Hi,
> >
> > I am trying to do a co-IP experiment on a HA tagged protein, and I
eluted
> > the protein with SDS buffer.  However, the protein level is very low
that
> > after I ran the eluate on a gel followed by silver staining, I couldn't
see
> > any band.  So now I am thinking combining all the eluate from multiple
> > experiments.  Is there a way that I can concentrate all these proteins?
> > Will TCA precipitation work in the presence of SDS?  Or is there any
kind of
> > concentrator that I can use?  Thank you very much!
> >
> >
> >
> >
>





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