I have used PBS to homogenize the tissues for testing Akt and phosphorylation of Akt using ELISA kit
The fact is that Akt was tested but pho-Akt showed nothing (the signal is zero in UV)
Is the lysis buffer the reason?
Or, I need to purify the homogenate with ultrafiltration to pre-concentrate the phosphorylation Akt? Looking forward to your answers...
Thank you very much
Did you include some phosphatase inhibitors to your sample buffer? Otherwise this enzymes can remove phosphate groups from other proteins
Aftab, I suppose that PMSF is a serine protease inhibitor, not a phosphoprotein phosphatase inhibitor. Anyway, both protease and phoshatase inhibitors are required for quantification.
Theoretically, all enzymes may continue working after the cell lysis. I hope that your protocol includes cooling during all steps of sample preparation...
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