Lipids off from Protein-Homogenate?

Newswatcher Newswatcher at sickkids.on.ca
Thu Jan 28 13:57:00 EST 1999


> > 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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