acetone precipitation of samples (for SDS PAGEs)
ckwen at expert.cc.purdue.edu
Mon Nov 18 09:43:01 EST 1996
David C. Logan (david.logan at plant-sciences.oxford.ac.uk) wrote:
: Sai Iyer wrote:
: >hey all,
: >i've posted this before, but i figured i'd try again to see if
: >there were any other ideas. i've been doing acetone precipitation to
: >remove salts from my samples prior to loading on sds gels. the basic
: >protocol is add 8 vol. of acetone to 1 vol. of protein sample --->
: >precipitate at -20 degrees for 4-6 hours ---> spin down for 20 min at
: >14,000g (max speed on centrifuge) at 4 degrees to pellet out the
: >precipitated protein ----> decant the supernatant and immediately
: >solubilize the pellet in sds loading buffer. the problem is see is
: >that i still get streaks in my gels. theoretically, acetone is an
: >uncharged organic solvent and so should not create any distortions in
: >the gel cuz
: >its not charged. so if there were any residual acetone left, that
: >shouldnt create the streaks...yet i still see streaks. anybody have
: >ideas on this? or other methods to remove salts from samples (i.e tca
: >precipitations etc) would also be welcome...thx...cheers....
I had problems with precipitated proteins, too. What I guess is that the
proteins I prepared were crude extract from the plant tissues, and, thus,
contained a lot of junk which could be polysaccharides and nucleic acids.
Acetone also precipitated those junks and denature them. The denatured
polysaccharides or other macromolecules were difficult to be dissolved
and gave problems during electrophoresis.
Just my opinions and wish to get correct answers.
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