> > I try to make 2D electrophoresis with rat brain tissue. Can somebody tell me
> > how i can get the the lipids off from my Protein homogenate, without
> > decreasing the amount of the proteins ?
>> Try a chloroform/methanol extraction (ratio 1:10 v/v). Add this to your
>sample,
> store at -70 degrees celsius (minimum of 1 hour). Centrifuge at ~14000g x 30
> min. Remove supernatent and dry sample (use a speedvac). Solubilise in 2D
> sample buffer as usual.
One concern that I would have with this procedure is that you will get
some protein aggregation as a result of the chloroform/methanol mixture
denaturing the protein. The method that we usually use in our lab is to
extra the protein homogenate 2 or 3 times with carbon tetrachloride.
CCl4 is almost completely non-miscible in water and therefore there is not
as much of a concern with it adding to protein denaturation. ;-)
Hope that this idea helps. ;-)
Best of Luck,
Scott Bukovac
bukovac at sickkids.on.ca
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