Western Blot and Inmunofluorescence contradictions - (Apr/28/2014 )
Technically there are several AA that can be phosphorilated according to Pubmed. If any of these phosphorilations take place only inside the nucleus (and inside the epitope) they could block the binding of the antibody, explaining why I don't see signal inside the nucleus by IF.
The problem I see with this is that asuming that my protein is phosphorilated at the nucleus and thus blocking the union of the antibody to the nuclear fraction of my protein, this should also happen with the cell-fractionation by WB since I'm using the same antibody for IF and WB?
ghernandez on Wed Apr 30 07:44:38 2014 said:
The problem I see with this is that assuming that my protein is phosphorylated at the nucleus and thus blocking the union of the antibody to the nuclear fraction of my protein, this should also happen with the cell-fractionation by WB since I'm using the same antibody for IF and WB?
a couple of ways that this might happen:
you may be detecting the cytoplasmic protein in the wb
and/or
phosphatases released by lysing and fractionation may be dephosphorylating the protein
mdfenko on Wed Apr 30 12:38:49 2014 said:
ghernandez on Wed Apr 30 07:44:38 2014 said:
The problem I see with this is that assuming that my protein is phosphorylated at the nucleus and thus blocking the union of the antibody to the nuclear fraction of my protein, this should also happen with the cell-fractionation by WB since I'm using the same antibody for IF and WB?
a couple of ways that this might happen:
you may be detecting the cytoplasmic protein in the wb
and/or
phosphatases released by lysing and fractionation may be dephosphorylating the protein
I doubt that's the first one since I'm detecting my protein on the chromatin but I'm not seeing the cytoplasm control (GADPH) there.
For the second one, I always use a cocktail of phosphatases inhibitors for RIPA buffer or any buffer where I'm working with proteins. But I suppose the cocktail coul not be working properly.