Description
Cholesterol Assay Kit (Colorimetric) (BA0084) (BA0084)
The Cholesterol Assay Kit (Colorimetric) (SKU: BA0084) offers a simple, direct and automation-ready procedure for measuring cholesterol. Cholesterol is a sterol and lipid present in cell membranes and transported in the bloodstream, and it plays important roles in membrane structure, hormone formation and cell signalling; elevated levels are associated with cardiovascular disease while low levels may be linked to depression, cancer and cerebral haemorrhage. This assay is based on cholesterol esterase hydrolysis of cholesterol esters to free cholesterol, followed by cholesterol dehydrogenase-catalysed 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 proportional to the cholesterol concentration in the sample.
| Product Name: | Cholesterol Assay Kit (Colorimetric) (BA0084) |
| SKU: | BA0084 |
| Detection Method: | Colorimetric |
| Detection Range: | 5 to 300 mg/dL |
| Sample Type: | Serum, plasma and other biological samples |
| Species Reactivity: | All |
| Assay Time: | 30 minutes at room temperature |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
Quantitative colorimetric determination of cholesterol at 340 nm. Cholesterol esters are hydrolysed to free cholesterol, which is converted to cholest-4-ene-3-one with the reduction of NAD to NADH; the NADH absorbance at 340 nm is proportional to cholesterol concentration.
- Sensitive and accurate; detection limit of 5 mg/dL and linearity up to 300 mg/dL
- Convenient room-temperature assay with no 37C heater required
- High-throughput; readily automated for thousands of samples per day
- Uses a single enzyme mix and NAD solution
- Direct assays of cholesterol in serum, plasma and other biological samples
- Pharmacology: effects of drugs on cholesterol metabolism
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Bring all reagents to room temperature prior to assay. Serum and plasma samples should be clear and free of turbidity; remove any precipitates by filtration or centrifugation. If not assayed immediately, samples can be stored at -20 to -80C for at least one year. |
| 2 | Standard curve. Prepare a 10-fold diluted standard by mixing 40 uL of the 300 mg/dL standard and 360 uL Assay Buffer, then dilute further in Assay Buffer as shown in the dilution table. Transfer 50 uL diluted standards into wells of the 96-well plate. |
| 3 | Samples. Dilute samples 10-fold (e.g. 10 uL sample with 90 uL Assay Buffer) and transfer 50 uL diluted sample into separate wells. |
| 4 | Prepare enough NAD solution by mixing, per reaction well, 40 uL Assay Buffer with 18 uL of the provided NAD Solution. Add 50 uL diluted NAD to the standard and sample wells, tap to mix, stand 5 minutes at room temperature and read background optical density at 340 nm (OD0). |
| 5 | Prepare enzyme mix by mixing, per reaction well, 10 uL Assay Buffer with 1 uL of the provided Enzyme Mix. Add 10 uL diluted enzyme mix per well and tap to mix thoroughly. |
| 6 | Incubate 30 minutes at room temperature and read OD30 at 340 nm. |
Subtract OD0 from OD30 for the standard and sample wells and use the dOD values to determine sample cholesterol concentration from the standard curve. Because both the standards and samples were diluted 10-fold, no additional dilution factor is required. If the sample OD exceeds that of the 300 mg/dL standard, dilute the sample in water, repeat the assay and multiply the result by the dilution factor.
| Component | Quantity | Storage |
| Assay Buffer | 20 mL | -20C |
| Enzyme Mix | 120 uL | -20C |
| NAD Solution | 2 x 1 mL | -20C |
| Standard (300 mg/dL cholesterol) | 1 mL | -20C |
