Dera all,
I'm searching for antibodies that detect phosphorelated form of protein and I found some antibodies that recognize both phosphorelated and unphosphorylated forms of proteins. I just want to know is it a good way to use this kind of product? If yes, does anyone know how to use it? I mean when I run the gel how can I recognize which form is detected?
Sure, depending on the quality and specifity of antibody this is a useful tool for simultaneous analyses of phosphorylated and total proteins.
I have used once an antibody that recognizes both phosphorylated and unphosphorylated form of the protein (antibody IRAK1 (D51G7) Rabbit mAb #4504 from cell signaling). It used to work very well because the protein auto-phosphorylates massively after stimulation, so its molecular weight increases after phosphorylation and the shift in the protein size will be thus detectable by WB.
In other cases, I don't believe that it will be a good idea to use an antibody that recognizes both forms especially if you are assessing the amount of protein phosphorylation, where you will need to normalise phospho-protien/total protein amount.
But again, it depends on the case (the protein, the aim of your experiment, etc.)
Depending on the desired protein, many companies have antibodies to phosphorylated and unphosporylated versions. Depending on the distance of where the epitopes are, it is possible to use both to identify active forms of phosphorylated antibodies using FRET.
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