Cholesterol Assay Kit

Catalog No. ABIN1000297

Product Details

Target: Cholesterol (CH)

Application: Biochemical Assay (BCA)

Sample Type: Plasma, Serum

Specificity: 5 mg/dL

Characteristics: Sensitive and accurate. Detection limit of 5 mg/dL, linearity up to 300 mg/dL cholesterol in 96-well plate assay.
Convenient. Room temperature assay. No 37°C heater is needed.
High-throughput. Can be readily automated as a high-throughput 96- well plate assay for thousands of samples per day.

Components: Assay Buffer: 20 mL. Enzyme Mix: 120 uL. NAD Solution: 2 x 1 mL. Standard: 1 mL 300mg/dL cholesterol.

Material not included: Pipetting (multi-channel) devices, clear bottom 96-well plate and plate reader.

Application Details

Application Notes: Direct Assays: cholesterol in serum, plasma, and other biological samples.
Pharmacology: effects of drugs on cholesterol metabolism.

Protocol: 1. Standard Curve. Prepare a 10-fold diluted standard (STD) by mixing 40 µL 300 mg/dL Standard and 360 µL Assay Buffer. Further dilute standard (STD) in Assay Buffer. Transfer 50 µL diluted standards into wells of the 96-well plate. Samples: dilute samples 10-fold (e.g. 10 µL sample with 90 µL Assay Buffer). Transfer 50 µL diluted sample in separate wells.
2. Prepare enough NAD solution in Assay Buffer : for each reaction well, mix 40 µL Assay Buffer with 18 µL the provided NAD Solution. Add 50 µL of diluted NAD to standards and sample wells. Tap plate to mix well. Let stand 5 min at room temperature. Read background optical density at 340nm (ODo).
3. Prepare enough enzyme mix : for each reaction well, mix 10 µL Assay Buffer with 1 µL provided Enzyme Mix. Add 10 µL diluted enzyme mix per well. Tap plate to mix thoroughly. Note: the enzyme mix may appear to be turbid, but will be clear after mixing into the reaction mixture.
4. Incubate 30 min at room temperature. Read OD30 at 340nm.
5. Calculation. Subtract OD0 from OD30 for the standard and sample wells. Use the OD values to determine sample cholesterol concentration from the standard curve. Note: since both the standards and samples were diluted 10-fold, no dilution factor is required. Note: if the sample OD value is higher than OD for the 300 mg/dL standard, dilute sample in water and repeat the assay. Multiply the results by the dilution factor.

Reagent Preparation:

Bring all reagents to room temperature prior to assay. Serum and plasma samples should be clear and free of turbidity or precipitates. If present, precipitates should be removed by filtration or centrifugation in a table centrifuge. If not assayed immediately, samples can be stored at -20 to -80°C for at least one year.

Restrictions: For Research Use only

Handling

Storage: -20 °C

References

Kang, Lee, Yi, Lee, Park, Park, Park, Choi: "Risk of cardiovascular disease is suppressed by dietary supplementation with protamine and chitooligosaccharide in Sprague-Dawley rats." in: Molecular medicine reports, Vol. 7, Issue 1, pp. 127-33, (2013) (PubMed).

Safwat, Yassin, Gamal El Din, Kassem: "Modulation of skeletal muscle performance and SERCA by exercise and adiponectin gene therapy in insulin-resistant rat." in: DNA and cell biology, Vol. 32, Issue 7, pp. 378-85, (2013) (PubMed).

Kang, Lee, Yi, Park, Lee, Park, Hwang, Park, Choi: "Modulation of lipid metabolism by mixtures of protamine and chitooligosaccharide through pancreatic lipase inhibitory activity in a rat model." in: Laboratory animal research, Vol. 28, Issue 1, pp. 31-8, (2012) (PubMed).

Seo, Ha, Kim: "α-Lipoic acid reduced weight gain and improved the lipid profile in rats fed with high fat diet." in: Nutrition research and practice, Vol. 6, Issue 3, pp. 195-200, (2012) (PubMed).

Schlotter, Matsumoto, Mangner, Schuler, Linke, Adams: "Regular exercise or changing diet does not influence aortic valve disease progression in LDLR deficient mice." in: PLoS ONE, Vol. 7, Issue 5, pp. e37298, (2012) (PubMed).

Rubinstein, Pelosi, Vedre, Kotaru, Abela: "Hypercholesterolemia and myocardial function evaluated via tissue doppler imaging." in: Cardiovascular ultrasound, Vol. 7, pp. 56, (2009) (PubMed).

Target Details

Target: Cholesterol (CH)

Alternative Name: Cholesterol (CH Products)

Background: Quantitative determination of cholesterol at 340nm.
Procedure: 40 min.

Cholesterol is a sterol and lipid present in the cell membranes, and is transported in the bloodstream of all animals. It is used to form cell membranes and hormones, and plays important roles in cell signaling processes. Elevated levels (hypercholesterolemia) have been associated with cardiovascular diseases such as atherosclerosis, whereas, low levels (hypocholesterolemia) may be linked to depression, cancer and cerebral hemorrhage. Simple, direct and automation-ready procedures for measuring cholesterol are very desirable. This Cholesterol Assay is based on cholesterol esterase hydrolysis of cholesterol esters to form free cholesterol and cholesterol dehydrogenase catalyzed conversion of cholesterol to cholest-4-ene- 3-one, in which NAD is reduced to NADH. The optical density of the formed NADH at 340 nm is directly proportionate to the cholesterol concentration in the sample.