I cannot detect the phosphorylation protein in my nuclei and cytoplasmic seperation samples, however, the whole protein could be detected well. I had add the protease inhibitors before the experiments all the time…… And the phosphorylation antibody also works well in whole cell lysis.
Can anyone help me to solve this problem?
Hi Yy Huang
Did you use phosphatase inhibitor cocktail? Did you use it during the whole partioning procedure? Did you detect the non phosphorylated protein?
Yy Huang
I had a problem like yours once. My protein of interest wasn't very abundant. I was only able to detect it using a very high amount of protein fraction (which made the gel look really ugly) or using immunoprecipitation (which gave a better result). If your antibody is suitable to immunoprecipitation you may give it a try.
Hi Yy,
Andre is absolutely right. You have to immunoprecipitate your protein of choice first, then run the gel and develop the blot using antibody against phosphorylated form of your target protein. Hope it will help.
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