Improved conditions for activity gel analysis of DNA polymerase catalytic polypeptides.
In a study of mouse DNA polymerase catalytic polypeptides using activity gel analysis, it was found that the sensitivity of detection of purified enzymes is markedly increased by addition of a heterogeneous mixture of proteins to the enzyme sample prior to electrophoresis (Karawya E., and Wilson, S.H. (1982) J. Biol. Chem. 257, 13,129-13,134). This modification and the use of a micromolar level of [32P]dNTP substrate are the basis of an improved activity gel assay for DNA polymerase catalytic polypeptides. This modified assay is several orders of magnitude more sensitive than the original procedure (Spanos, A., Sedgwick, S.G., Yarranton, G.T., Hubscher, U., and Banks, G.R. (1981) Nucl. Acids Res. 9, 1825-1839), and it enables measurement of two reference enzymes, calf beta-polymerase and Escherichia coli DNA polymerase I large fragment, in the picogram range. Further, it was found that it is essential to survey different lots of sodium dodecyl sulfate to identify those which enable high enzyme activity signals after renaturation.
D000818:Animals; D001345:Autoradiography; D004247:DNA / Q000378:metabolism; D004256:DNA Polymerase I / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005069:Evaluation Studies as Topic; D006358:Hot Temperature; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D011506:Proteins / Q000378:metabolism
D011506:Proteins; D004247:DNA; D004256:DNA Polymerase I