When I analyze cytoplasmic protein detection by FACS, the result is negative. However the result is positive when analyzing by Western Blot showing a band with the right M.W. What is the possible reason?
Antibody working concentration is low.
Cell membrane was not penetrated sufficiently.
Antibody incubation time is too short.
Detection of FACS excitation wavelength is not consistent with fluorescence-labeled antibody excitation wavelength.