HELP! Triton X114 blues...

Steve Dunn dunnsm at bbsrc.ac.uk
Wed Mar 5 12:40:13 EST 1997


Dear netters,

I've been dabbling with Triton X114 phase-partitioning of my microsomal
fraction.  After washing the detergent-rich phase a few times with PBS,
I (following a procedure in Methods In Enzymology...) precipitate the
protein with 10 volumes of ice-cold acetone.  This, according to my method,
is supposed to get rid of the detergent so that it doesn't interfere with
SDS-PAGE gels. After pelleting the protein and removing all the
acetone, I resuspend the pellet in 5%SDS and loading buffer for SDS-PAGE.
However, after staining the gel with coomassie, there's an artifactual
smear obscuring my protein bands which I understand is symptomatic of
X114 contamination.  Even after processing the acetone pellet through a
chloroform/MeOH cleanup (Wessel & Flugge, Anal. Biochem.,1984) the smear
persists! If I run just SDS-solubilised microsomes, there's no smear.
Any ideas how to successfully combine X114 extraction with SDS-PAGE??
All suggestions gratefully received...

Steve Dunn
dunnsm at bbsrc.ac.uk





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