Cholesterol is a lipid present in the cell membranes of eukaryotes and circulates in the blood stream. It is used in the biosynthesis of hormones, and plays an important role in cell signaling processes. Cholesterol exists as a free acid, as well as, in the esterified form as cholesteryl esters. Elevated levels of cholesterol are indicated in atherosclerosis and heart disease, and are the subject of large amount of research focused on cholesterol metabolism. Quantitative determination of cholesterol in experimental samples is central to this research.
Cell Technology’s Total Cholesterol kit is a simple one step assay which can be used either as a Fluorimetric or Colorimetric assay to help detect the level of total cholesterol in samples.
- Detection of Total Cholesterol in Biological Samples.
- Easy to Use -Simple ONE STEP assay.
- Used to study the effect of drugs on Cholesterol Metabolism.
- Highly Sensitive – Detects up to 200nM of Cholesterol.
- Versatile – Fluorimetric or Colorimetric readout in a 96 well format.
Cell Technology’s Cholesterol assay kit provides a simple, one-step fluorimetric or colorimetric method for the determination of total cholesterol in serum and plasma samples. The assay is based on an enzyme-coupled reaction that detects both free cholesterol and cholesterol esters. Cholesterol esters are hydrolyzed by cholesterol esterase into cholesterol, which is then oxidized by cholesterol oxidase to yield hydrogen peroxide and cholest-4-en-3-one (ketone). The hydrogen peroxide then reacts with the cholesterol probe (detection reagent) in a 1:1 stoichiometry to produce the stable fluorescent product
↓ Cholesterol esterase
↓ Cholesterol Oxidase
H2O2 + Cholest-4-en-3-one
λmax 570nm. λ ex/em 535/585nm.
Colorimetric assay can be read on a spectrophotometer at 570nm.
Fluorescence is measured at excitation 530nm and emission 585nm.
Figure 1. Cholesterol standard curve was generated using fluorimetric detection: excitation 535nm and emission 585nm. R2 value=0.9977. Incubation time= 1 hr at Room temperature. Standard curve range 0.15625 µM to 10µM.
Figure 2. Cholesterol standard curve generated using colorimetric detection: absorbance read-out at 570nm. R2 =0.9993. Incubation time=30 minutes. Standard curve range 1.25µM to 80µM.
Figure 3. demonstrates the quantitation of total cholesterol in mg/dL in animal sera