membrane fraction extraction for PKC - (Jun/13/2005 )

I am having trouble in PKC translocation experiments.
after stimulation with PMA, protein kinase C beta (PKC) seems to translocate to other place since PKCbeta in the cytosol decreased significantly. But I can not find it in the membrane fraction In my experiments. I did it according to paper published in J Biol Chem. 2005 Mar 18;280(11):9870-8. Epub 2005 Jan 4. It titled "Translocation of diacylglycerol kinase theta from cytosol to plasma membrane in response to activation of G protein-coupled receptors and protein kinase C."

I wonder the protocol has some problem or I am wrongly doing some performance in my experiment.

appreciate your help!

---

Two questions:

Did you check the cytosolic fraction to be sure it wasn't there?

What method are you using to detect you PKC.

---

Thanks for your replying.
Yes, I checked the cytosolic fraction and only little PKC was there compared with the control.
I used Western blotting to detect PKC .

---