PHOSPHOR IMAGE-ANALYSIS OF HUMAN P53 PROTEIN ISOFORMS

Phosphor imaging was evaluated for detection, quantitation and resolut ion of multiphosphorylated protein isoforms separated by two-dimension al gel electrophoresis. A nuclear phosphoprotein, p53, was isolated by immunoprecipitation after biosynthetic labeling with S-35, P-32 or P- 33 in cultured human cells. Of the three radionuclides, S-35 was the m ost sensitive in detection after a 1-week exposure, although shorter e xposure times were effective. In dividing cells, 11 S-35-labeled isofo rms were found, of which 10 were phosphorylated by P-33 and P-32. Expo sure of phosphonuclides for one half-life showed that P-33 radiolabeli ng produced better resolution among isoforms than P-32 but was less se nsitive in detection. Volume integration showed phosphorylated isoform s comprised from 1% to 25% of total isoform signal. The relative phosp horylation of each p53 isoform wa estimated by normalizing P-33 or P-3 2 isoform volumes with the corresponding S-35 volume and showed progre ssive phosphorylation of acidic isoforms. Additionally, phosphor imagi ng capably detected quantitative changes among individual isoforms aft er experimental modulation of the isoform pattern by serum deprivation . The described electrophoretic isolation and quantitation procedures should find general application in discerning active and inactive phos phoisoforms for eventual identification.