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.
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 .