I am trying to immunoprecipitate a myc-tagged ABC transporter from yeast
(S. pombe). My C-terminal myc tagged protein works great for western
blots and immunofluorescence but I can't seem to get it to IP from yeast
extracts using fairly standard protocols. My protein is solubilized
fairly efficiently with SDS - I can't release it from the membrane with
any other detergent yet (I've tried BM's detergent kit to try a variety).
In the final IP the SDS conc. is around 0.1%. I'm wondering if my
hydrophobic membrane protein is folding in such a way to hide the myc-tag
and make it difficult for the monoclonal antibody to find it. I can't, for
various reasons, move the tag to another location. Does anybody know of a
source of a myc-tagged protein or Human leukaemia nuclear extracts I can
use as a positive control for my IP's? Also does anyone have any advice on
optimising IP's for such proteins? Any advice appreciated!
Sonya Clark
PGEC-USDA
Albany CA
saclark at nature.berkeley.edu
