I would like to know if anybody is familiar with phosphatase activity assay and more precisely with PP2A activity in cells treated with OP449. First of all, I have already shown that neuroblastoma are sensitive to OP499, an inhibitor of SET oncogene, which activates PP2A. And now I want to prove that cell death is mediated by PP2A reactivation upon OP449 treatment. Unfortunately, I am not able to get an activity in cells treated with this compound as the untreated control already shows an activity similar to the treated samples, and I can´t find out after many expereiments what the problem. I optimized the immunoprecipitation. I decontaminated my samples from any phosphate that would ihhibit the phosphatase activity. All buffers that I am using are free of phosphate. Samples are stored at -80°C and always handled on ice. I tried several time points and different concentrations of the compound and still no good results. I am wondering where do I go wrong. I am using for this assay the PP2A immunoprecipitation kit from Millipore.
Thank you for your responses
In the absence of critical experimental details such as treatment time length and antibody specificity to precipitate PP2A and the assay conditions for the phosphatase activity, it is harder to identify the problem. Therefore, it is likely that storing samples at -80 degree C could alter the activity in treated samples without protease inhibitor that prevent cleavage of proteins in lysate. Therefore, all steps need to be carried out without freezing the samples and at 4 degree C. If this does not help, share your observations again to seek additional help tips.
Thank you Dikaver for your response
Actually, I have tried 30 min, 4h, 8h and 24h time points
the samples are treated with protease inhibitor as well. Moreover,
I have tried already to carry the experiments without freezing the samples, but there is no improvement at all. That´s why I am asking if somebody knows how samples should be handled to do not alter the activity in my treated samples because I have the impression that there is a problem of phosphatase activity stability here...
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