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Total Cholesterol and Cholesteryl Ester Colorimetric Assay Kit II

based on 9 citations in multiple journalsTotal Cholesterol and Cholesteryl Ester Colorimetric Assay Kit II94.2 4
Stable, Sensitive Assay
Catalog #: K623

In stock

$405.00

Product Details

Cat # +Size K623-100
Size 100 assays
Detection Method Absorbance (450 nm)
Species Reactivity Mammalian
Applications N/A
Features & Benefits • Simple procedure; takes ~1 hour
• Fast and convenient
• Kit contains the necessary reagents for measurement of cholesterol in various biological samples
Kit Components • Cholesterol Assay Buffer
• Substrate Mix (lyophilized)
• Enzyme Mix (lyophilized)
• Esterase (lyophilized)
• Cholesterol Standard (2 µg/µl)
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.

Details

Cholesterol is an essential molecule in all animal life. It has been involved in both normal development and diseases. The Cholesterol/Cholesteryl Ester Quantitation Kit provides a simple method for sensitive quantification of free cholesterol, cholesteryl esters, or both using a colorimetric method. Most of the cholesterol in blood is in the form of cholesteryl ester. These esters can be hydrolyzed to free cholesterol under the appropriate conditions. In the assay, free cholesterol is oxidized by cholesterol dehydrogenase to generate NADH which reacts with a sensitive probe resulting in strong absorbance at 450 nm. The assay can detect free or total cholesterol depending upon whether esterase is utilized to hydrolyze cholesterol esters present. Cholesteryl ester can be determined by subtracting the value of free cholesterol from the total cholesterol (cholesterol plus cholesteryl esters). The probe in this kit is more stable, sensitive and specific. The assay can tolerate interferences from various samples significantly.


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What is the concentration of free cholesterol in the Cholesterol Standard (or ratio between free and ester-bound cholesterol)?
The detailed composition of the standard is proprietary information. But most of the cholesterol in serum exists as cholesterol ester. Our standard has similar cholesterol to cholesterol ester ratio as serum.
Can a standard curve for free cholestrol be prepared if only free cholesterol is to be assayed in the samples?
Our standard has similar cholesterol to cholesterol ester ratio as serum. The free cholesterol in the standard is oxidized directly by cholesterol dehydrogenase to generate NADH which reacts with the probe. When esterase is added, the cholesterol esters are broken down into cholesterol and then the same reaction proceeds to give color. If only free cholesterol is to be assayed in the sample, a std curve can be prepared without esterase. Upon inclusion of the esterase, the total (free + ester form) is quantified. Hence if the difference of Total-free cholesterol is calculated, that is the amount of cholesterol ester in the sample.
Can Ergosterol in samples interfere in this assay?
Ergosterol is found in yeast and fungal cell membranes. It should not be detected by this assay. We have not tested this per se but most of BioVision products are optimized to be compatible with mammalian sources. The first step of this detection process is specific for cholesterol and cholesterol esters. Inefficient conversion of Ergosterol might be possible but this can be subtracted by using a parallel control.
To extract the lipids Folch's method was used and the lipids were resuspended in isopropanol. Are these samples compatible with K623?
Samples resuspended in Isopropanol is not likely to work with this kit. The reaction mechanism is designed to be in aqueous solution.
How long should samples be air-dried ?
Typically up to 1 hour is enough to air- dry at 50C to remove chloroform. The goal to remove as much chloroform at this step as possible. Over-drying should be avoided. Upon overdrying it becomes difficult to dissolve in assay buffer.
Can individual components of this kit be purchased separately?
Yes, any of the kit's components can be purchased separately without having to buy the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can frozen cells/tissues be used with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can the dried extracted lipids be stored at -20C or -80C to be assayed later?
It is preferred that the cells/samples be stored at -80C before lipid extraction. Storing the extracted lipids could lead to aggregation and other issues like lipid extract sticking to the tube walls. This could lead to under-estimation of the amount of cholesterol in the sample.
When using this kit with adipose tissue, several layers were seen after the 15,000g spin instead of a single homogeneous organic phase. Which of the layer/s should be collected?
The organic phase may seem to have multiple layers, so collect all. This should be a max of 200 µl. Since it contains a high concentration of chloroform and isopropyl alcohol, which evaporate fast, it could take 1- a couple of hrs to dry out at 50°C. The time can be inceased based on the sample.
Sundaram, M. et al., (2017) he apolipoprotein C-III (Gln38Lys) variant associated with human hypertriglyceridemia is a gain-of-function mutation, Journal of Lipid Research, Sep.2017, jlr-M077313
Lin, NN et al., (2017) Bacillus Subtilis-Fermented Red Bean (Red Bean Natto) Reduces Hyperlipidemia Levels in Hamsters Fed an Atherogenic Diet, J Food Biochemistry, 2017, 41(1): online
Liu et al., Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia. Dis. Model. Mech., Aug 2015; 8: 989 - 998.
Xu et al., Rutaecarpine suppresses atherosclerosis in ApoE–/– mice through upregulating ABCA1 and SR-BI within RCT. J. Lipid Res., Aug 2014; 55: 1634 - 1647.
Watanabe et al., Lipocalin 2 binds to membrane phosphatidylethanolamine to induce lipid raft movement in a PKA-dependent manner and modulates sperm maturation. Development, May 2014; 141: 2157 - 2164.
For more citations of this product click here