Can total and phosphorylated protein be analyzed in parallel?
For BD Phosflow applications, we recommend comparing the basal phospho-protein level from an unstimulated sample (the negative control) against the phospho-protein content of the stimulated sample, rather than comparing the content of total protein and phospho-protein together in the same sample. In some cases, however, investigators may be interested in analyzing the total content of a specific protein (for example, when comparing different human tumor samples for which a negative equivalent cannot be obtained). We have antibodies against total proteins (ERK, p38, AKT, Stat, etc) but have not validated these for BD Phosflow protocols. In general, antibodies recognizing epitopes against a total protein are unlikely to recognize a specific phospho-epitope. Therefore, any standard intracellular staining protocol should be suitable with the total protein antibody. If the antibody is suitable for immunofluorescence (IF), it will likely be compatible for flow cytometry. However, please keep
