EnzyChrom™ AF Cholesterol Assay Kit

Protocol SDS

Application

• For quantitative determination of cholesterol and evaluation of drug effects on cholesterol metabolism.

Key Features

• Sensitive and accurate. Linear detection range in 96-well plate: 0.1 to 10 mg/dL cholesterol for colorimetric assays and 0.02 to 2 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.

Method

• OD570nm, or FL530/585nm

Samples

• Serum, plasma, etc

Species

• All

Procedure

• 40 min

Size

• 100 tests

Detection Limit

• OD, FL: 1, 0.2 mg/dL

Shelf Life

• 12 months

More Details

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. BioAssay Systems EnzyChrom™ 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 λex/em = 530/585nm is directly proportional to the total cholesterol concentration in the sample.

I would like to know whether egg yolk can be used for such a kit. If this doesn’t work, can you suggest us another protocol, please?

Egg yolk cholesterol extraction protocol:

1. Prepare extraction buffer with 5 vol isopropanol, 2 vol water, 2 vol Triton X-100.

In a 1.5 mL Eppendorf tube, mix 50 µL yolk with 450 µL of the extraction buffer. Vortex for at least 30 sec.

3. Centrifuge 4 min at 14,000rpm on a table centrifuge.

4. Remove supernatant and perform cholesterol assay.

What is the principle of the E2CH-100 cholesterol assay?

Cholesterol esterase cleaves esters into cholesterol, which is oxidized to form H₂O₂ in the presence of cholesterol oxidase. The produced H₂O₂ is measured with a specific dye Ampliflu in the presence of a peroxidase.

What substances are known to interfere with cholesterol assay?

Reducing agents such as ascorbic acid and bilirubin interfere with peroxidase-dependent cholesterol determinations.

Hernandez-Corroto, E. et al. (2020). Sustainable extraction of proteins and bioactive substances from pomegranate peel (Punica granatum L.) using pressurized liquids and deep eutectic solvents.Innovative Food Science & Emerging Technologies. 60: 102314. Assay: Cholesterol in pomegranate peel extracts.

Wilson, L. J. (2020). Integrating upstream and downstream process development strategies for mammalian cell derived therapeutic antibodies (Master’s thesis, UCL (University College London), 2020). 1-227. Assay: Cholesterol in hamster cells.

Attah, F. A., Asaleye, C. M., Omisore, A. D., Kolawole, B. A., Aderibigbe, A. S., & Alo, M. (2019). Relationship between sonographically measured median nerve cross-sectional area and presence of peripheral neuropathy in diabetic subjects. World journal of diabetes, 10(1), 47. Assay: Cholesterol in human blood.

Kumar, A., Pandita, S., Anand Laxmi, N., Bhakat, M., & Mohanty, T. K. (2018). Effects of prostasomes on functional parameters of fresh and cryopreserved-thawed spermatozoa of crossbred Karan Fries (KF) bulls. Indian. J Anim. Res. Assay: Cholesterol in bulls plasma.

Chappuis, E., Morel-Depeisse, F., Bariohay, B., & Roux, J. (2017). Alpha-galacto-oligosaccharides at low dose improve liver steatosis in a high-fat diet mouse model. Molecules, 22(10), 1725. Assay: Cholesterol in mice plasma.

Gallagher, A. J., Skubel, R. A., Pethybridge, H. R., & Hammerschlag, N. (2017). Energy metabolism in mobile, wild-sampled sharks inferred by plasma lipids. Conservation physiology 5(1):cox002. Assay: Cholesterol in shark blood.

Sinha, P. B., Tesfaye, D., Rings, F., Hossien, M., Hoelker, M., Held, E. & Salilew-Wondim, D. (2017). MicroRNA-130b is involved in bovine granulosa and cumulus cells function, oocyte maturation and blastocyst formation. Journal of ovarian research, 10(1), 37. Assay: Cholesterol in bovine cells.

Raij, L., Tian, R., Wong, J. S., He, J. C., & Campbell, K. N. (2016). Podocyte injury: the role of proteinuria, urinary plasminogen, and oxidative stress. American Journal of Physiology-Renal Physiology, 311(6), F1308-F1317. Assay: Cholesterol in human podocyte.

Guevara-Arauza, J.C., et al. (2011). Biofunctional activity of tortillas and bars enhanced with nopal. Preliminary assessment of functional effect after intake on the oxidative status in healthy volunteers. Chem Cent J 5(1):10. Assay: Cholesterol in human plasma.

Stoll, C., et al. (2011). Liposomes alter thermal phase behavior and composition of red blood cell membranes. Biochim Biophys Acta 1808(1):474-81. Assay: Cholesterol in human red blood cells.

Uddin, M.J., et al. (2011). Detection of quantitative trait loci affecting serum cholesterol, LDL, HDL, and triglyceride in pigs. BMC Genet 12:62. Assay: Cholesterol in pig serum.

Waheed, M.M., et al. (2011). Some Biochemical Characteristics and Preservation of Epididymal Camel Spermatozoa (Camelus dromedarius). Theriogenology 76(6):1126-33. Assay: Cholesterol in Camelus dromedarius epididymal fluid.

Ponda, MP et al (2010). Moderate kidney disease inhibits atherosclerosis regression. Atherosclerosis 210(1):57-62. Assay: Cholesterol in mouse blood.

Rub A et al (2009). Cholesterol depletion associated with Leishmania major infection alters macrophage CD40 signalosome composition and effector function. Nat Immunol. 10(3):273-80. Assay: Cholesterol in mouse macrophage, membrane.

Lee, SM et al (2008).GCG-rich tea catechins are effective in lowering cholesterol and triglyceride concentrations in hyperlipidemic rats. Lipids 43(5): 419-429. Assay: cholesterol in rat blood.

To find more recent publications, please click here.

If you or your labs do not have the equipment or scientists necessary to run this assay, BioAssay Systems can perform the service for you.

– Fast turnaround
– Quality data
– Low cost

Please email or call 1-510-782-9988 x 2 to discuss your projects.