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Problem with Hsp72 - (Nov/04/2004 )

Hello, my name is Sergio, and I have a problem with the protein Hsp72 in human smooth muscle cell. I dont see any difference between TNF treated (50 nanograms/mL, for six hours) and non-treated control cells. I have test heat shock (42 C for 30 min, and then 6 hours at 37 C). These parameter I have tested, can be found in many reports in the literature. I have test diferents time of serum starvation and without serum starvation (control and treated cells). The lysis buffer contains HEPES, NaCl, NaF, Na3VO4, B-glicerophosphate, aprotinin, leupeptin, pepstatin, PMSF, Benzamidine, EDTA, EGTA and DTT. The concentrations are the same as other messages posted here.
The cell line have been bought from Clonetics. It is not an explant. The experiments have been developed until passage 10 using the special medium for optimal growth as recomended by Clonetics.
I charge 30 micrograms of protein, this ought to be enough because I can see an only band in the membrane, so the electroforesis and the western blot I think is not the problem.
I have tested different passages, cell lines, times of incubation and I think I am getting mad...
Please, any idea?

-serglom-

Hi Sergio

I have a lot of experience with heat shocking and WB for Hsp72. I think the problem is that your heat shock is not sufficient. The best conditions to induce Hsps is 42 C for 60 min in a water bath. Your 6 hours recovery at 37 C should be fine then. Also you might want to check that your thermometer is working correctly.

-cconcannon-

QUOTE (cconcannon @ Nov 11 2004, 04:20 AM)
Hi Sergio

I have a lot of experience with heat shocking and WB for Hsp72.  I think the problem is that your heat shock is not sufficient.  The best conditions to induce Hsps is 42 C for 60 min in a water bath.  Your 6 hours recovery at 37 C should be fine then.  Also you might want to check that your thermometer is working correctly.

Thanks for your reply, ill test those conditions
Yours,
Sergio

-serglom-