Is it necessary to add phosphatase inhibitors prior to insulin or what precaution needs to be taken before freezing the desired tissue?
We've got good western blot results (looking at phosphorylation of downstream insulin signalling proteins) in snap frozen mouse liver without adding phosphatase inhibitors during homogenisation on ice, although it surely depends on the exact protein of interest.
Indeed, snap freezing using liquid nitrogen is the way to go. You just have to be careful with the protein extraction process by using proteases and phosphatases inhibitors.
Make very sure that you process the samples quickly and the condition should always be maintained cold untill you denature the protein. Have you tried using the PI3K lysis buffer???
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