This might be a very odd question to you. I am planning to detect pRB protein in formalin-fixed paraffin-embedded materials. Unfortunately we have antibodies (Ab) directed to all forms of pRB; meaning hyperphosphorylated and hypophosphorylated forms.
To find out which form I am looking at after staining, I thought about protein phosphatase treatment of the coups prior to pRB Ab incubation. Does this make any sense? Perhaps you have tried this or have a better idea about it; e.g., buying phospho-specific Abs?
are there three groups of RBs? Unphosphorylated, hypo-p and hyper-p? Phosphatase treatment will only give you unphosphorylated RB. For phosphatase treatment, I would use lamda-phosphatase (from New England Biolabs). It is potent and cheap.
I do not understand how would a phospho-antibody work identically on both hyperphosphorylated target and hypophosphorylated target. Site-specific phospho-antibodies are the way to go.
Thanks Tung for your response. I am also new to this. Its is believed that hypo-p pRB is the active form by binding to E2F and inhibiting its function. Hyper-p pRB on the other hand is the inactive form of pRB, meaning E2F is released and can function properly during cell cycling.
I have looked around and found an Ab, offered by the company cell signaling technology, that does recognize phosphorylation site-specific pRB at ser807/811. So I will try this option in combination with lamda-phosphatase as you suggested.
We hope this will answer our ideas!
Found Ab detecting ser807/811 phospho-pRB (see picture for a double IFA stain, Red=Ki67; Green=Phospho-pRB; Blue=nuclei). Now trying to dephosphorylate this residues.....
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