IP is usually used to confirm protein phosphorylation, but if the antibody used has a weak affinity to the target protein, can we extend the incubation time to overnight if the cell lysate is provided with PhosSTOP (from Roche) to a final concentration at 1X?
This is a very good question which I also had once. If the antibody has weak affinity towards phospho protein you can try loading more protein in the gel for electrophoresis. Adding a phosphatase inhibitor cocktail is a good idea. You can try even adding beta-glycerophosphate as a pseudo substrate for the phosphatase which will compete with the phospho protein and will g ive a better phospho signal in western blot.
I would strongly recommend including phosphatase inhibitors in lysis buffer and buffers used for washing Protein A/G sepharose, if immunoprecipitating.
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