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
