Gen5 version required: 1.0 or higher
Basis for the Assay:
Catalase enzyme catalyzes the conversion of hydrogen peroxide to water. Amplex® Red, when present of with horseradish peroxidase enzyme, reacts with H2O2 in a 1:1 stiochiometry to produce resorufin, a red fluorescent compound, which has an absorption and fluorescence emission maxima of 563 nm and 587 nm respectively. Therefore the presence of catalase would be expected to reduce the amount of fluorescence produced from a constant amount of HRP and H2O2 present in a well. The amount of catalase present is proportional to the change (reduction) in fluorescence generated.
The protocol calls for a fluorescence measurement using a 540/35 excitation filter and a 590/20 emission filter. The data reduction uses a transformation to subtract the value of each well from the mean of the 0 mU/ml catalase standard (STD1). This difference in fluorescence is then plotted against known catalase concentrations in order to provide a calibration curve.
The experiment data file contains all of the fluorescence determinations for a catalase enzyme activity determination using Amplex Red as the substrate.
Plate configuration assumes that samples will be run in duplicate. Included is a standard curve of catalase enzyme activity (0 to 1000 U/ml) as defined by Invitrogen’s Catalase detection kit. The plate includes 8 standard concentrations, but no blank or controls wells. Thus, 40 samples can be run on a microplate in duplicate, in addition to the standard curve.
The report is configured to provide the (1) raw fluorescence, (2) standard curve, (3) calculated catalase concentrations and statistics of unknown samples.