AF Cholesterol Assay Kit

Catalog No. ABIN1000281

Product Details

Target: AF Cholesterol

Application: Biochemical Assay (BCA)

Sample Type: Plasma, Serum

Characteristics: Sensitive and accurate. Linear detection range in 96-well plate: 1 to 100 mg/dL cholesterol for colorimetric assays and 0.2 to 10 mg/dL for fluorimetric assays.
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. Dye Reagent: 120µL. Standard: 1 mL 300 mg/dL cholesterol.

Material not included: Pipetting (multi-channel) devices, 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 100 mg/dL standard (STD) by mixing 15 µL 300 mg/dL Standard and 435 µL Assay Buffer. Further dilute standard (STD) in Assay Buffer as shown below. Transfer 50 µL diluted standards into wells of a clear 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. For each reaction well, mix 55 µL Assay Buffer with 1 µL Enzyme Mix and 1 µL Dye Reagent. Add 50 µL of this Working Reagent to each standard and sample well. Tap plate to mix well.
3. Incubate 30 min at room temperature. Read OD at 570 nm. Note: if the Sample OD is higher than the Standard OD at 100 mg/dL, dilute sample in assay buffer and repeat the assay. Multiply result by the dilution factor.

Reagent Preparation:

Important: 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. If not assayed immediately, samples can be stored at - 20 to -80°C for at least one year.

Calculation of Results:

Subtract blank OD (water, #8) from the standard OD values and plot the OD against standard concentrations. Note: since both the standards and samples were diluted 10-fold, no dilution factor is required.

Restrictions: For Research Use only

Handling

Storage: -20 °C

References

Guevara-Arauza, Ornelas Paz, Mendoza, Guerra, Paz Maldonado, González: "Biofunctional activity of tortillas and bars enhanced with nopal. Preliminary assessment of functional effect after intake on the oxidative status in healthy volunteers." in: Chemistry Central journal, Vol. 5, Issue 1, pp. 10, (2011) (PubMed).

Uddin, Duy, Cinar, Tesfaye, Tholen, Juengst, Looft, Schellander: "Detection of quantitative trait loci affecting serum cholesterol, LDL, HDL, and triglyceride in pigs." in: BMC genetics, Vol. 12, pp. 62, (2011) (PubMed).

Waheed, Al-Eknah, El-Bahr: "Some biochemical characteristics and preservation of epididymal camel spermatozoa (Camelus dromedarius)." in: Theriogenology, Vol. 76, Issue 6, pp. 1126-33, (2011) (PubMed).

Ponda, Barash, Feig, Fisher, Skolnik: "Moderate kidney disease inhibits atherosclerosis regression." in: Atherosclerosis, Vol. 210, Issue 1, pp. 57-62, (2010) (PubMed).

Stoll, Stadnick, Kollas, Holovati, Glasmacher, Acker, Wolkers: "Liposomes alter thermal phase behavior and composition of red blood cell membranes." in: Biochimica et biophysica acta, Vol. 1808, Issue 1, pp. 474-81, (2010) (PubMed).

Rub, Dey, Jadhav, Kamat, Chakkaramakkil, Majumdar, Mukhopadhyaya, Saha: "Cholesterol depletion associated with Leishmania major infection alters macrophage CD40 signalosome composition and effector function." in: Nature immunology, Vol. 10, Issue 3, pp. 273-80, (2009) (PubMed).

Lee, Kim, Kim, Shin, Baik: "GCG-rich tea catechins are effective in lowering cholesterol and triglyceride concentrations in hyperlipidemic rats." in: Lipids, Vol. 43, Issue 5, pp. 419-29, (2008) (PubMed).

Target Details

Target: AF Cholesterol

Background: Quantitative determination of cholesterol by colorimetric (570nm) or fluorometric (530nm/590nm) methods.
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 uses a single Working Reagent that combines cholesterol ester hydrolysis, oxidation and color reaction in one step. The color intensity of the reaction product at 570nm or fluorescence intensity at gamma em/ex = 585/530nm is directly proportional to total cholesterol concentration in the sample.