Cholesterol Assay Kit, BioAssay(TM), Colorimetric

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. \n\nSimple, direct and automation-ready procedures for measuring cholesterol are very desirable. 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.\n\nApplications:\nDirect Assays: cholesterol in serum, plasma, and other biological samples.\nPharmacology: effects of drugs on cholesterol metabolism.\n\nKey Features:\nSensitive and accurate. Detection limit of 5mg/dL, linearity up to 300mg/dL cholesterol in 96-well plate assay.\nConvenient. Room temperature assay. No 37 degrees C heater is needed.\nHigh-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.\n\nKit Contents: (100 tests in 96-well plates)\nAssay Buffer: 20ml Enzyme Mix: 120uL\nNAD Solution: 2 x 1ml Standard: 1ml 300mg/dL cholesterol\nStorage conditions. Store reagents at -20 degrees C. Shelf life: 6 months after receipt.\nPrecautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.\n\nProcedures:\nImportant: 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 degrees C for at least one year.\n1. Standard Curve. Prepare a 10-fold diluted standard (STD) by mixing 40ul 300mg/dL Standard and 360ul Assay Buffer. Further dilute standard (STD) in Assay Buffer as shown below.\nNo STD+Assay Buffer Vol (ul) 10 x Conc. (mg/dL)\n1 100ul+0ul 100 300\n2 80ul+20ul 100 240\n3 60ul+40ul 100 180\n4 40ul+60ul 100 120\n5 30ul+70ul 100 90\n6 20ul+80ul 100 60\n7 10ul+90ul 100 30\n8 0ul+100ul 100 0\nTransfer 50ul diluted standards into wells of the 96-well plate. Samples: dilute samples 10-fold (e.g. 10ul sample with 90ul Assay Buffer). Transfer 50ul diluted sample in separate wells.\n2. Prepare enough NAD solution in Assay Buffer as follows: for each reaction well, mix 40ul Assay Buffer with 18ul the provided NAD Solution. Add 50ul 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).\n3. Prepare enough enzyme mix as follows: for each reaction well, mix 10ul Assay Buffer with 1ul provided Enzyme Mix. Add 10ul 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.\n4. Incubate 30 min at room temperature. Read OD30 at 340nm.\n5. Calculation:. Subtract OD0 from OD30 for the standard and sample wells. Use the DOD 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.\nNote: 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.\n\nMaterials Required, But Not Provided:\nPipetting (multi-channel) devices, clear bottom 96-well plate and plate reader.\n\nExamples:\nSamples were run in duplicate according to the standard procedure.\nThe cholesterol concentrations (mg/dL) were 105

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SPECIFICATIONS

Catalog Number

C5045-10

Size

1Kit

References

1. Lee, S.M. et al (2008). GCG-Rich Tea Catechins are Effective in Lowering Cholesterol and Triglyceride Concentrations in Hyperlipidemic Rats. Lipids 43: 419-429.\n2. Khan, M.A. et al (2009). Statins impair CD1d-mediated antigen presentation through the inhibition of prenylation. J Immunol 182(8): 4744-4750.\n3. Mellado, M. et al (2008). Rough agave flowers as a potential feed resource for growing goats. Rangeland Ecol Manage 61: 640- 646.