I've been analyzing for a few months a protein both by western-blot and by inmunofluorescence and I'm having what it seems to be contradictory results. Let me explain:
-When I generate protein extracts by cell-fractionation (cytoplasm-nucleoplasm-chromatin) and analyze them by western blot i detect my protein at all the 3 fractions. As a control for the fractionation I use GADPH for cytolplasm (mostly cytoplasm with a minor staining at nucleoplasm but I've heard that there's always minor contamination) and ORC2 for nuclei (mostly chromatin but also in nucleoplasm).
-When performing an IF protocol (formaldehid fixation) I only see staining at the cytoplasm.
-Also the antibody I use for WB and IF it also works for inmunoprecipitation so we can rule out that the antibody is not recognizing the native protein.
I dont know how to explain it. Maybe i have to modify the IF protocol or the cell-fractionation?
Thank you in advance for your time and answers,
No need to double post - other one deleted. Bob.