Analysis on heterogeneity of squamous cell carcinoma antigen by two-dimensional electrophoresis

Squamous cell carcinoma (SCC) antigen was separated by two-dimensional electrophoresis combined with immunoblotting into four spots: spot 1 with pI 6.4 and 44.5 kDa, spot 2 with pI 6.3 and 44.5 kDa, spot 3 with pI 6.0 and 44.5 kDa, and spot 4 with pI 5.9 and 45 kDa. In cancer and noncancerous tissues, it was common that spot 1 was the largest spot. In noncancerous tissues, spot 3 was the smallest spot and spot 2 was stained as densely as spot 4. In cancer tissues, however, spot 4 was apparently smaller than spot 2 and 3. Also, spots 2 and 3 in cancer tissues were larger than those in noncancerous tissues. When SCC antigen was treated with alkaline phosphatase prior to isoelectric focusing (IEF), spot 4 disappeared from the immunoblotting pattern. When the SCC antigen was treated with alkaline phosphatase after IEF, spot 4 changed its molecular weight to the same weight as that of the other three spots. These results strongly suggest that spot 4 is phosphorylated SCC antigen.