I am just getting started in this area and was interested in looking at the protein expression of the phosphorylated form of a transcription factor, both in the nucleus and cytosol fractions. Could anyone help with a protocol or hints for the nuclear/cytosol extraction of phosphorylated proteins from a cell culture sample? I will probably lyse with NP-40, but unsure of the inhibitors, especially the phosphatase inhibitors I should add (what concentrations, at what step/what buffers). Thanks so much for your help and time! Diane
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