ballal_sv at hotmail.com
Tue Sep 3 22:42:25 EST 2002
There seems to be nothing much wrong with your protocol. Only thing I
guess is 1000 rpm to pellet the ppt is rather low!!!??
I guess the storage of your protein in cold itself will cause a lot of
them to precipitate. I you can actually add acetone and then freeze
the same. ( Won't take much extra time and should not matter if you
keep it overnight in -80 C freezer).
Also giving an extra wash with 100% Acetone helps to remove remaining
1.) Bring protein solution to 80% acetone using HPLC-grade.(4 volumes)
2.) Incubate at -20°C overnight or in dry ice for 2-3 hours. Don't cut
3.) Centrifuge 10 minutes at 4°C and carefully remove supernatant.
4.) Wash pellet gently with two aliquots of 100% acetone at -20°C.
5.) Dry sample briefly under vacuum and store sealed at -20°C. ( you
can also dry for longer time at RT)
Best fo Luck
Indus Biotherapeutics Ltd.
Michael Witty <mw132 at mole.bio.cam.ac.uk> wrote in message news:<Pine.SGI.4.33.0209021216470.1455074-100000 at mole.bio.cam.ac.uk>...
> 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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