Protein precipitation

Christopher Cilley cdcilley at OXYTRICHA.MIT.EDU
Wed Apr 13 18:37:27 EST 1994


I've been using this for years...  (Thanks to Lilia Babe' who sent it when I
left it behind.)

TCA/DOC precipitation of proteins

Solns:  TCA/DOC solution-
        4mg/ml DOC in 100% TCA (100% TCA made as per Maniatis)
        (DOC is Deoxycholic Acid)

Protocol:
        Add 1/10 volume of TCA/DOC solution, vortex
        15' on ice
        Spin 14,000g (ufuge on max) 10'
        Aspirate supernatant
        Add 1ml -20degreeC acetone, vortex briefly
        Spin 14,000g 10'
        Aspirate supernatant
        Speed-vac to dry (about 15')
        Add 1:1 water:4X Laemmli, vortex, 95degreeC 10' and load

Notes:
        You can use less DOC in the TCA/DOC soln. if it seems to much to
                resuspend in the volumes you are using.
        Use less TCA/DOC (down to 1/20 volume if there is a lot of protein
                in your sample.
        You can leave on ice (or 4degreeC) overnight if you like.  Slightly
                shorter times are ok too.
        If the solution is yellow after adding the Laemmli add 0.5-1 ul of
                2M Tris, not pH'd to bring the pH up to where it should be.
        The precipitation doesn't work so well if the [Ammonium Sulfate] is
                greater than 20%.  You'll need to dilute it to <20% before
                precipitating.

Luck,
-- 

        - Christopher

"People who don't fight the unjust condemnation of others 
 but internalize it are, Miss Manners admits, badly in need of 
 lessons in self-esteem."       -Judith Martin (aka Miss Manners)

cdcilley at oxytricha.mit.edu




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