Promega, J3191, Cholesterol/Cholesterol Ester-Glo™ Assay

How the Assay Works
The Cholesterol/Cholesterol Ester-Glo™ Assay measures cholesterol using a cholesterol dehydrogenase that links the presence of cholesterol to the production of NADH and the activation of a proluciferin that produces light with luciferase. In the presence of NADH, Reductase enzymatically reduces a proluciferin Reductase Substrate to luciferin. Luciferin is detected in a luciferase reaction using Ultra-Glo™ Luciferase and ATP, and the amount of light produced is proportional to the amount of cholesterol in the sample.
For measuring Cholesterol Ester, the assay includes a cholesterol esterase that will remove the fatty acid from cholesterol esters to produce one molecule of cholesterol per molecule of ester. The amount of cholesterol esters is determined from the difference of cholesterol measured in the absence (free cholesterol) and presence (total cholesterol) of esterase.
Simple Protocol is Compatible with Diverse Sample Types
Cholesterol Detection in Mouse Liver Tissue
Mouse liver tissue was homogenized and diluted in Lysis Solution. A cholesterol standard spike of 10μM was added to an aliquot of diluted sample, then all samples and standard were assayed for free cholesterol. The change between standard and background is the same as the change between sample + standard and sample, indicating that the tissue homogenate did not interfere with quantitation of the cholesterol standard.
Cholesterol Detection in Human Lipoproteins
Fractions of lipoproteins contain different amounts of cholesterol and cholesterol esters. The cholesterol detection assay can be used to quantify total cholesterol levels, free cholesterol levels and calculate the cholesterol ester levels as the difference between the two values.