A quantitative microtiter plate nuclease assay based on ethidium/DNA fluorescence.
A microtiter plate assay was developed to quantitate the nuclease activity of the extracellular Serratia marcescens endonuclease under different buffer conditions. Substrate cleavage was followed as decrease in ethidium/DNA fluorescence using a uv-transilluminator and a video documentation system. Time courses of DNA cleavage were recorded and cleavage rates determined very precisely within a factor of 1.2. The assay has a linear dynamic range covering three orders of magnitude of nuclease activity and can be carried out very quickly within a few minutes. It can also be used with RNA as substrate. With appropriate modifications it should be possible to adapt this assay for other enzymatic reactions which are accompanied by changes in absorbance or fluorescence.
Friedhoff
P
P
Institut für Biochemie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, Giessen, D-35392, Germany.
Matzen
S E
SE
Meiss
G
G
Pingoud
A
A
D000818:Animals; D002712:Chlorides; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004706:Endodeoxyribonucleases / Q000737:chemistry*; D004722:Endoribonucleases / Q000737:chemistry*; D004996:Ethidium / Q000737:chemistry*; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride; D017895:Manganese Compounds; D011485:Protein Binding; D012313:RNA / Q000032:analysis / Q000737:chemistry
D002712:Chlorides; D017895:Manganese Compounds; D015636:Magnesium Chloride; D012313:RNA; D004247:DNA; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; C021835:Serratia marcescens nuclease; D004996:Ethidium; C025340:manganese chloride