Detection of enzymatic activities in sodium dodecyl sulfate-polyacrylamide gels: DNA polymerases as model enzymes.
Recent techniques for detecting the catalytic activity of enzymes in sodium dodecyl sulfate (SDS)-polyacrylamide gels have been hampered by lack of reproducibility associated with variability in commercial SDS preparations. Simple expedients which facilitate reproducible detection of DNA polymerase activity and which appear to be widely applicable to detection of other enzymes are reported here. It was observed that reproducibility of a reported procedure for DNA polymerase detection (Spanos, A., Sedgwick, S. G., Yarranton, G. T., Hübscher, U., and Banks, G. R. (1981) Nucl. Acids Res. 9, 1825-1839) depends on the SDS used for electrophoresis, and that sensitivity is markedly reduced if currently available SDS is substituted for the discontinued product specified by Spanos et al. A modified procedure yielding sensitivity with contemporary commercial SDS, which exceeds the sensitivity observed when using the protocol and the SDS originally specified, is described. The modifications employed, which presumably promote renaturation of enzymes, are (1) embedding fibrinogen in gels and (2) washing detergent from gels with aqueous isopropanol after electrophoresis. These expedients permit detection of picogram amounts of Escherichia coli DNA polymerase 1 and its Klenow fragment and nanogram amounts of calf thymus alpha and rat liver (Novikoff hepatoma) beta polymerases. Finally, it is shown that sensitivity of DNA polymerase detection is reduced by lipophilic contaminants in contemporary commercial SDS, and that the expedients employed here mitigate the deleterious effect of these impurities.
D000180:Acrylic Resins / Q000032:analysis*; D000818:Animals; D002417:Cattle; D004259:DNA-Directed DNA Polymerase / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D051381:Rats; D012967:Sodium Dodecyl Sulfate
D000180:Acrylic Resins; C016680:polyacrylamide gels; D012967:Sodium Dodecyl Sulfate; D004259:DNA-Directed DNA Polymerase