Acetone precipitation

Vayuputra vayuputra369 at hotmail.com
Sun Sep 1 21:12:02 EST 2002


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







More information about the Proteins mailing list