On 5 Mar 1997 17:36:21 GMT, dunnsm at bbsrc.ac.uk (Steve Dunn) wrote:
>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.ukDear Steve
Triton is know to be able to form peroxides in solution , which might
partially react with your protein and therefore cause this smear.
Check if your Triton is expired, store in the dark(!) , use reducing
agents to reduce the peroxides(e.g.:DTT) ...
Hope this helps
Martin
