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Re: protein G IP



Dear Erno -

Here's some ideas, which you probably thought of, but here they are anyway.
1.  Make sure the protein G is well blocked with BSA.
2. Try a substrate that's more specific for your kinase than MBP, which is
a substrate for several kinases.
3.  Try a different kind of bead to pull down your monoclonal - such as
anti-mouse IgG beads.
4.  There are usually contaminating kinase activities in "negative control"
IPs.  The trick is to maximize the specific signal and minimize the
non-specific background.  It may not be possible to eliminate the
non-specific background entirely.  For example, perhaps your antibody kills
the activity of the kinase it binds to.  If you tried a different antibody,
you might boost the signal to such an extent that the background would
become (relatively) insignificant.

Good luck,

Lee Bardwell


 >Dear All,
>We found kinase activity -phosphorylation of MBP- in our negative control
>experiments which consist of immunoprecipitation using protein G and a
>hippocampal lysate only. No antibody was involved. Preclearing the lysate
>with protein G didnot remove this kinase activity. Protein G alone didnot
>have this kinase activity. Thus, it looks as if protein G is capable to
>immunoprecipitate  kinase activity in hippocampal lysate.  Does anyone
>recognize this phenomenon? How to solve this problem? (how to do IP with
>our kinase antibody?). We are desperate for suggestions!!
>Thanks, Erno



*****************************************************
Dr. Lee Bardwell, Asst. Prof.
Dept. of Developmental & Cell Biology
University of California at Irvine
5207 Biological Sciences II
Irvine, CA  92697-2300
bardwell@uci.edu
office:	949 824 6902
lab:	949 824 6908
fax:	949 824 4709
web site: http://darwin.bio.uci.edu/~bardwell
*****************************************************