"Active" two-dimensional electrophoresis of rat liver DNA-polymerase alpha

The approach of so-called active gel analysis was used to determine the position and appearance of the catalytic subunit of rat liver DNA polymerase alpha on a two-dimensional (2-D) electrophoretic map. In this case a polyacrylamide gel containing DNA was used for the second dimension. DNA presence does not change the 2-D protein pattern but makes it possible to conduct a polymerase reaction directly in the gel after separation. A crude extract of rat liver nuclei was used for analysis. The extract is quickly isolated and contains mainly DNA polymerase alpha activity. It was shown that this enzyme restores its activity after 2-D electrophoresis and sodium dodecyl sulfate (SDS) elution. After polymerase reaction with labeled dNTPs and autoradiography, the catalytic polypeptide or, rather, polypeptide cluster is revealed as chains of spots (possibly because of the presence of different hydrolyzed and phosphorylated forms). These spots are located on the 2-D electrophoretic map in the region corresponding to molecular masses of 160, 140, and 130 kDa and pI 5.5-6.2.