immunopptn

Rob Jordan Jordan at mbcrr.harvard.edu
Fri May 19 10:58:59 EST 1995


In article <3pfeko$n41 at lyra.csx.cam.ac.uk>, rw200 at cus.cam.ac.uk (R.
Woodward) wrote:

> Dear all
> I am having problems with immunoprecipitation of invitro translated
products (from a Promega TNT coupled transcription translation system). I
get product from the translation mixture of the correct size(as judged by
SDS-PAGE fluorography)but I am unable to immunoprecipitate any of the
product.  My protein is FLAG epitope tagged and I have varified that the
FLAG DNA sequence in the cDNA is present and o.k. also the batch of
antiserum I have recognises another FLAG construct by western blotECL. I
am using v.
> mild conditions for my immunopptn (NET-Gel buffer) an antidody dilution
of 1:3000 (incubation time + temp have been varied) and formalin fixed
S.aureus (Sigma) unfortunately I have no positive controls. Any ideas.   
My protein is an ion channel subunit so  do I need to add pancreatic
microsomes to my mix in order to get correct folding for recognition by my
anti FLAG antibody. Any thoughts or experience would be appreciated.

Your epitope may be masked when your protein acheives its native
conformation.. You could try moving the epitope tag to a new location with
in your protein or perhaps add denaturant before you immunoprecipitate.

Good Luck,

Rob J.

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