Hello,
I am attempting to detect a phosphorylated protein using immunofluorescence. Should I include a phosphatase inhibitor in my blocking buffer? Will a protein's phosphorylation status change from paraformaldehyde fixation or Triton X-100 membrane permeabilization?
Thank you.
After you've fixed your sample (with PFA4%), proteins are fixed, so there will be no protease/phosphatase activity (assuming you really fixed samples properly).
About your question whether the protein status will change due to TX-100 or PFA, if you are working with membrane proteins, overfixation or over-permeabilization may result in dissolving of some of the membrane, and it will be hard for you to get a signal (but that's not due to phosphatase activity, it's because your protein of interest went somewhere).
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