I made an phospho specific antibody. I checked this antibody using KI cell lines. there is pan protein but no signal with phospho antibody. i thought it means my phospho antibody is specific. then i made an KO and reconstituted KO with WT and phospho mutant protein. they are based on tet on system. i checked these reconstituted proteins with phospho antibody. i found sometimes there were signals in mutant cells sometimes not. i am a little confused now. is it possible that when i used too much protein that phospho antibody also recognized surrounding amino acid sequence? does anyone have experience with this?
I used to do a lot of this in a past life. So your knock-in (e.g. Ser-to-Ala mutant) is nice support of phospho-specificity, but sometimes the substitution is enough to block binding (i.e. you're testing for serine-specificity). It's better to modulate the phosphorylation itself. If there is not a physiological treatment that completely modulates phosphorylation, you can dephosphorylate the site completely with a phosphatase such as lambda ppase, by lysing cells directly in Ppase buffer and incubation with the enzyme. For a positive control, again a known physiological stimulus is good, but you can try treating cells with a ppase inhibitor such as calyculin a and then lyse them as you normally would.
Here are a couple examples of this kind of phosphospecificity testing:
https://www.cellsignal.com/products/primary-antibodies/phospho-rb-ser608-antibody/2181
https://www.cellsignal.com/products/primary-antibodies/phospho-atr-ser428-antibody/2853
https://www.cellsignal.com/products/primary-antibodies/phospho-p53-ser33-antibody/2526
As Andreas mentioned, antibody-antigen binding might depend on the relative concentrations of both, and of course polyclonal antibodies might be a mixture of phosphospecific, phosphoselective, and general antibodies.
Good luck.
thanks all guys for your suggestions. i found there were bands when i incubated with primary antibody at room temperature but bands were gone at 4.
You can also make an antibody to non phosphorylated sequence to differentiate phosphorylated and non phosphorylated.
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