Cholesterol (Amplex Red)
November 27, 2012Download
Related Products: Gen5 for Detection
Gen5 version required: 1.0 or higher
Basis for the Assay:
Cholesterol esterase is used to hydrolyze any cholesterol ester present to free cholesterol. Free cholesterol is converted to cholest-4-ene-3-one ketone product by the action of cholesterol oxidase and in doing so generates hydrogen peroxide (H2O2). Amplex red reagent and H2O2 are converted to resorufin and water in a one-to-one stiochiometry by horseradish peroxidase. Resorufin is a highly fluorescent compound with absorption maxima of 563 nm and a peak emission wavelength of 587 nm. The amount of fluorescence produced is directly proportional to the amount of total cholesterol (free and esterified) present in the sample.
The protocol calls for a fluorescence measurement using a 540/35 excitation filter and a 590/20 emission filter.
The experiment data file contains all of the fluorescence determinations for a Cholesterol determination using Invitrogen’s Cholesterol determination kit.
Plate configuration assumes that samples will be run in duplicate. Included is a standard curve of Cholesterol concentration (0 to 10 μM) as defined by Invitrogen’s Cholesterol determination kit. The plate includes 8 standard concentrations, but no blank or control 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 cholesterol concentrations and statistics of unknown samples.