Acetone pellets and protein insolubility
rmiller16 at nc.rr.com
Mon May 21 21:26:40 EST 2001
in article 3B08CB2D.B0DA9B1D at med.monash.edu.au, Paul Cullen at
Paul.Cullen at med.monash.edu.au wrote on 5/21/01 4:00 AM:
> Hi everyone,
> I am acetone precipitating proteins from the aqueous and detergent
> phases of a Triton X114 extraction. The problem is that the pellets if
> allowed to dry become hard like rocks and insoluble in normal 1D sample
> buffer for protein gels. I want to store the precipitated proteins for
> several weeks before I go to a specialised proteomics facility. I am
> worried if I freeze dry these pellets that they won't be of any use when
> i get to the proteomics facility. These are the ideas I have come up with:
> 1) Leave the proteins in acetone solution.
> - I've herd this can also make the pellet impossible to resuspend.
> 2) Try to resuspend the samples in PBS and then freeze dry or store at -70
> - I can't resuspend them in sample buffer because the proteomics place
> where i'm going has proprietary detergent solutions which they will
> resuspend them in.
> - I can't imagine the detergent phase proteins would go into a PBS solution.
> 3) Don't resuspend them in PBS, just layer it over the top of the
> pellets and store at -70.
> Please help or give any suggestions that you can.
Usually the problem with insoluble acetone powders is all of the water was
not removed during the acetone precipitation step. The solution to this
problem is to not skimp on the amount of acetone used. If you have been
using 100 ml of acetone, increase it to 200 ml and be sure the acetone is
dry. After the powder is formed, spread it out on 3 MM paper and work it
back and forth with a spatula to get as much of the residual acetone out to
the precipitate as possible as fast as possible. This procedure results in
an acetone powder that will easily go into solution in virtually any buffer.
An old hand at making acetone powders for enzyme preps,
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