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Total Cholesterol and Cholesteryl Ester Colorimetric/Fluorometric Assay Kit

based on 71 citations in multiple journalsTotal Cholesterol and Cholesteryl Ester Colorimetric/Fluorometric Assay Kit715 5
Catalog #: K603

In stock

$425.00

Product Details

Cat # +Size K603-100
Size 100 assays
Detection Method Absorbance (570 nm) or Fluorescence (Ex/Em 535/587 nm)
Species Reactivity Mammalian
Applications The assay detects total cholesterol (cholesterol and cholesteryl esters) in the presence of cholesterol esterase or free cholesterol in the absence of cholesterol esterase in the reaction.
Features & Benefits • Simple procedure; takes ~60 minutes
• Fast and convenient
Kit Components • Cholesterol Assay Buffer
• Cholesterol Probe (in DMSO, anhydrous)
• Enzyme Mix (lyophilized)
• Cholesterol 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

The Cholesterol/Cholesteryl Ester Quantitation Kit provides a simple method for sensitive quantification of free cholesterol, cholesteryl esters, or both by colorimetric or fluorometric methods. Majority of the cholesterol in blood is in the form of cholesteryl esters which can be hydrolyzed to cholesterol by cholesterol esterase. Cholesterol is then oxidized by cholesterol oxidase to yield H₂O₂which reacts with a sensitive cholesterol probe to produce color (λmax = 570 nm) and fluorescence (Ex/Em = 535/587 nm). The assay detects total cholesterol (cholesterol and cholesteryl esters) in the presence of cholesterol esterase or free cholesterol in the absence of cholesterol esterase in the reaction. Cholesteryl ester can be determined by subtracting the value of free cholesterol from the total (cholesterol plus cholesteryl esters).


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The enzyme does not fully dissolve in 220ul of Assay Buffer. Will this affect the results?
The enzyme solution is likely super-saturated and hence some material is insoluble. We have tested the assay with 100% solubilized enzyme and 50% solubilized enzyme and the results were identical. The enzyme solution can be warmed up briefly for 1-2 minutes at 37C to help solubilize more enzyme.
The probe changes to red color after dissolving. Does this affect the assay?
The probe changing to red color after dissolving in DMSO should not affect the quality of the assay. The probe will always be pink to red and the extent of redness depends on the level of oxidation. However, a pinkish/mild red color should not affect the assay significantly.
To measure free cholesterol and total cholesterol, are two separate sets of standard curves needed?
No. Only one standard curve after adding the cholesterol esterase should be produced.
In serum both esterified and non-esterified cholesterol is detectable. From tissue homogenates only the non-esterified version is detected. Why?
When tissues are homogenized, cellular enzymes including Esterases are released. These enzymes hydrolyze the esterified cholesterol to non-esterified cholesterol.
Can EDTA be used for blood collection for this assay?
Yes, EDTA will be fine.
Can this kit measure free or protein-bound cholesterol?
This kit can measure both free and apolipoprotein-bound forms of cholesterol.
What is the detailed principle of measurement?
Cholesterol esterase hydrolyzes cholesterol ester into free cholesterol. Then cholesterol oxidase in the enzyme mix oxidizes the free cholesterol to generate H2O2. H2O2 then interacts with the cholesterol probe to generate color/fluorescence.
Can this kit be used with tissue samples, e.g. liver?
Yes, tissue samples can be used with this kit. It is critical to homogenize the tissue in chloroform:Isopropanol: NP-40 for effective extraction of lipids from the sample. Please follow the instructions on the Datasheet for further details.
Are the enzyme mixes in K603 and K623 the same?
No. Although both kits measure cholsterol/cholesterol ester, the enzyme mixes contain different components and cannot be swapped between K603 and K623.
Can the Absorbance for this kit be read at 620nm instead of 570nm?
All spectrophotometers have a window within which they are equally accurate. Typically this window is +/-20 to +/-40nm. 620nm seems a bit far-stretched for 570nm recommended wavelength for this assay but I would suggest checking the instrument specifications.
Can frozen tissue be used with this kit?
Although not ideal, flash-frozen tissue or cells stored at -80C can be used. It is important to remember that results may vary between fresh and frozen samples.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Can individual components of this kit be purchased?
Yes, any of the kit's components can be purchased without having to buy the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can alternate buffers for sample preparation (cell lysis, sample dilutions etc) be used?
Our assay buffers are optimized for the reactions in this assay. They not only contain some detergents for efficient lysis of your cells, but also contain some proprietary components required for the detection reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Is it essential to make a standard curve for every expt, or is one curve/kit enough?
Yes, we strongly recommend you measure the standards every time you do an experiment. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
Zhang P et al., (2017) Beraprost sodium, a prostacyclin analogue, reduces fructose-induced hepatocellular steatosis in mice and in vitro via the microRNA-200a and SIRT1 signaling pathway, Metabolism, 2017, 73:9-21
Tse, Margaret Chui Ling et al. (2017) Tumor Necrosis Factor a Promotes Phosphoinositide 3-Kinase Enhancer A and Amp-Activated Protein Kinase Interaction to Suppress Lipid Oxidation in Skeletal Muscle, Diabetes. 2017 Jul;66(7):1858-1870.
Huang, Er-Wen et al. (2016) Endophilin-A2-mediated increase in scavenger receptor expression contributes to macrophage-derived foam cell formation, Atherosclerosis. 2016 Nov;254:133-141.
Soh, Serena Ying et al. (2016) NKT Cell Hyporesponsiveness Leads to Unrestrained Accumulation of Marginal Zone B Cells in Hypercholesterolemic Apolipoprotein E–Deficient Mice, J Immunol. 2016 Nov 15;197(10):3894-3904.
Yamamoto et al., Vascular endothelial cell membranes differentiate between stretch and shear stress through transitions in their lipid phases.  Am J Physiol Heart Circ Physiol, Oct 2015; 309: H1178 - H1185.
For more citations of this product click here
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