immunopptn

R. Woodward rw200 at cus.cam.ac.uk
Thu May 18 07:30:16 EST 1995


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.
Robert



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