Acetone precipitation

Michael Witty mw132 at mole.bio.cam.ac.uk
Mon Sep 2 06:17:35 EST 2002


On Sun, 1 Sep 2002, Vayuputra wrote:

> Hi,
>
> I'm trying to isolate a protein from fish embryos and am facing some
> problems.  My constraints are the starting material and the amount of
> endogenous protein.  The aim is to concentrate all protein so that I may
> assay it using Western analysis.  Any help would be greatly appreciated.
> Here is what I have been trying to do -
>
> 1.  homogenize the embryos in buffer (Hepes buffer 7.4, 150mM NaCl, EGTA and
> PMSF).
> 2.  centrifuge (~7500 g) to get rid of debris.
> 3.  take supernatant and put in -80 degrees C O/N (this step is only for
> time considerations.  prior to freezing the supernatant looks clear but once
> it is thawed I find lots of 'stuff'.  I don't know if storing the
> supernatant at -80 is bad).
> 4.  add acetone (upto 80%) and put at -80 degrees for 2-3 hours.
> 5.  spin at 1000 rpm for 20 minutes.
> 6.  pour off the acetone and dry the pellet (in speed vac).
>
> I try to dissolve this pellet in my loading buffer (SDS, 2-mercaptoethanol,
> bromophenol blue, etc) but I just can't.  I make sure the pellet is as dry
> as possible (to remove any possibility of residual acetone).
>
> Is there anything I'm doing wrong?  Are there any modifications to this
> protocol?
>
> Thanks a ton.
> Vayuputra

Dear Vayuputra,
              everything depends on what you want to do with the protein
and the assay you are going to use detect it.  Can you tell us more about
this?  Regards, Mike.




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