[Histonet] re:Abs against phosphoproteins

From:"Carl Hobbs"

I agree with Andrea. Sure, fixation ( and don't forget pwax processing) may 
well destroy/mask your protein irreversibly. Such that no amount of Ag 
retrieval will work. Always is a "luck of the draw" situation. Also, if the 
Ab is raised against a peptide, there is no guarantee that it will recognise 
the native protein, either.
The only way to confirm that your pwax- negative staining is "real", would 
be to use T/C cells that have been, for eg, irradiated, then fixed and 
stained for your p-Histone. Then, you would make a pellet of another plate 
of irradiated- fixed cells and process that to pwax. If the Ab is negative 
on these cells, whatever pretreatment you do, then it ain't gonna work on 
pwax sections, full stop. If you must use archival material, then try to 
find another Ab, raised for the same protein. I'm sure others will make more 
positive comments/ give alternative suggestions. Try asking here too.... 
Best wishes

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