Cholesterol Assay Kit, BioAssay™, Colorimetric

Cat# C5045-10-96T

Size : 96Tests

Marca : US Biological

Contatta il distributore locale :


Telefono : +1 850 650 7790


Shipping Temp
Dry Ice

Storage Temp
-20°C

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. 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.

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

Key Features:
Sensitive and accurate. Detection limit of 5mg/dL, linearity up to 300mg/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.

Kit Contents: (100 tests in 96-well plates)
Assay Buffer: 20ml Enzyme Mix: 120uL
NAD Solution: 2 x 1ml Standard: 1ml 300mg/dL cholesterol
Storage conditions. Store reagents at -20°C. Shelf life: 6 months after receipt.
Precautions: 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.

Procedures:
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 in a table centrifuge. If not assayed immediately, samples can be stored at -20 to -80°C for at least one year.
1. 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.
No STD+Assay Buffer Vol (ul) 10 x Conc. (mg/dL)
1 100ul+0ul 100 300
2 80ul+20ul 100 240
3 60ul+40ul 100 180
4 40ul+60ul 100 120
5 30ul+70ul 100 90
6 20ul+80ul 100 60
7 10ul+90ul 100 30
8 0ul+100ul 100 0
Transfer 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.
2. 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).
3. 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.
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 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.
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.

Materials Required, But Not Provided:
Pipetting (multi-channel) devices, clear bottom 96-well plate and plate reader.

Examples:
Samples were run in duplicate according to the standard procedure.
The cholesterol concentrations (mg/dL) were 105 ± 3 for a human serum, 155 ± 11 for human plasma, 157 ± 2 for a bovine serum, 68 ± 2 for a rat serum, 129 ± 3 for a mouse serum, 123 ± 2 for a goat serum sample.

Important Note
This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.