PKC translocation - how to detect PKC translocation (Jun/10/2005 )

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.

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Did you do a Western on Whole cell lysate to see if the overall level remains constant?
When Separating a cell into fractions, keep each fraction and run it on a gel to ensure you haven't lost your protein of interest in a particular fraction.

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thank you so much!

I did not do a Western on Whole cell lysate and this time I will do. You know I only give cell stimulation for 5 minutes. The overall level of PKC should not change.
yes, I did, but I can not find my interest protein only in the membrane fraction.

Hi all,

appreciate your help! any more suggestions?

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