mw132 at mole.bio.cam.ac.uk
Mon Sep 2 06:17:35 EST 2002
On Sun, 1 Sep 2002, Vayuputra wrote:
> 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
> 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
> Thanks a ton.
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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