Total Cholesterol(TC) Colorimetric Assay Kit(COD-PAP Method)

K086

48T(44S)/ 96T(92S)

Microplate reader(495-525 nm)

Universal

Quantitative detection of total cholesterol (TC) content in serum, plasma, tissue samples.

2-8°C for 6 months

See kit label

For research use only.

Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as to obtain sufficient amount of reagents.

Total cholesterol(TC) includes free cholesterol and cholesterol esters. Cholesterol ester can be hydrolyzed by cholesterol esterase(CE) to produce cholesterol and free fatty acid. Cholesterol is oxidized by cholesterol oxidase to produce △4-cholestenone and hydrogen peroxide. In the presence of 4-aminoamylpyridine and phenol, hydrogen peroxide catalyze peroxidase to form red quinone compounds of benzoquinone imine phenizone. The color depth of the generated quinone is directly proportional to the cholesterol content.

1.Microplate reader(495-525 nm)

2.96-well plate

3.Precision Micropipettor and clean disposable tips

4.Clean EP tubes

5.absolute ethanol, Normal saline (0.9% NaCl), Double distilled water

6.Benchtop centrifuge

7.Constant temperature water bath/incubator

Sample requirements: No reducing substances, such as ascorbic acid and glutathione, can be added to the sample.

Serum and plasma: detect directly.

Tissue sample: According to the tissue mass (g) : absolute ethanol volume (mL) 1:5 ~10 ratio (it is recommended to weigh about 0.1g tissue, add 1mL absolute ethanol), the mixture is homogenized in ice bath. 10000g, centrifuge at 4℃ for 10min, and take the supernatant on ice to be measured.

Before the formal test, 2-3 samples with significant differences should be selected and diluted into different concentrations for pre-test. According to the results of the pre-experiment and the linear range of this kit: 0.29-25.85 mmol/L, the recommended dilution factor for different samples is as follows (for reference only):

Note: The diluent of serum(plasma) is normal saline (0.9% NaCl). The diluent of tissue Sample is absolute ethanol.

Equilibrate all reagents to room temperature before use.

1.Standard well: Take 2.5 μL of standard to the wells.

Sample well: Take 2.5 μL of sample to the wells.

Blank well: Take 2.5 μL of double-distilled water to the wells.

2.Add 250 μL of Enzyme Solution to each well and mix fully.

3.Cover the plate sealer and incubate at 37 °C for 15 min. Measure the OD value of each well with microplate reader at 510 nm.

Note:

ΔA1: OD_sample-OD_blank

ΔA2: OD_standard-OD_blank

f: Dilution factor of sample before tested.

c: the concentration of standard, 5.17 mmol/L.

m: the weight of tissue sample, g.

V: the volume of the homogenate of tissue samples, mL.

Standard curve: (The standard curve is provided as below for reference only.)

1.This assay kit is for Research Use Only. Please read the instructions carefully and adjust the instruments before the experiments. Please follow the instructions strictly during the experiments. Protection methods must be taken by wearing lab coat and latex gloves.

2.The detection range of the kit is not equivalent to the concentration range of the substance to be measured in the sample. If the concentration of substance is not within the detection range exactly, an extra dilution or concentration should be taken for the sample. It is recommended to take a pre-test if your sample is not listed in the instruction book.

3.The experimental results are closely related to the situation of reagents, operations, environment and so on. Fine Test will guarantee the quality of the kits only, and NOT be responsible for the sample consumption caused by using the assay kits. It is better to calculate the possible usage of sample and reserve sufficient samples before use.