In situ detection of DNA-metabolizing enzymes following polyacrylamide gel electrophoresis.
We have presented several protocols for producing an in situ activity gel that allows detection of various DNA-metabolizing enzymes. Both nondenaturing polyacrylamide and SDS-polyacrylamide activity gel electrophoresis procedures were detailed. Combining the use of defined [32P]DNA substrates with product analysis, these procedures detected a wide spectrum of enzymatic activities. The ability to detect 7 different catalytic activities of 15 different enzymes provides encouragement for expanded applications. It is hoped that others will find this technique applicable for detecting these enzymes and other activities in different biological systems. The modification of DNA in situ and the creation of intermediate substrates within activity gels should prove extremely useful for dissecting the enzymatic steps of DNA replication, repair, recombination, and restriction, as well as the metabolic pathways of other nucleic acids.
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
M J
MJ
Longley
D W
DW
Mosbaugh
D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004256:DNA Polymerase I / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D013379:Substrate Specificity
D004279:DNA, Viral; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D004247:DNA; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V