Overview

  • Product name

    Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation
    See all Cholesterol/Cholesteryl Ester kits
  • Detection method

    Colorimetric/Fluorometric
  • Sample type

    Cell culture supernatant, Urine, Serum, Plasma, Other biological fluids, Tissue Extracts
  • Assay type

    Quantitative
  • Assay time

    1h 00m
  • Product overview

    Cholesterol/ Cholesteryl Ester Assay Kit ab65359 provides a simple method for sensitive quantification of total cholesterol, free cholesterol, and cholesteryl esters, by colorimetric or fluorometric methods.


    The majority of cholesterol in blood is in the form of cholesteryl esters. In the cholesterol assay protocol, these are hydrolyzed to cholesterol by cholesterol esterase. Cholesterol is then oxidized by cholesterol oxidase to yield H2O2 which 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) when cholesterol esterase is included in the reaction, or free cholesterol when it is not included. The amount of cholesteryl ester can be determined by subtracting the value of free cholesterol from the total (cholesterol plus cholesteryl esters).


    Cholesterol assay protocol summary:
    - add samples and standards to wells
    - add total cholesterol reaction mix (with esterase) or free cholesterol reaction mix (without esterase) and incubate for 60 min at 37ºC
    - analyze with microplate reader

  • Notes

    Other cholesterol assay kits include:
    HDL and LDL/VLDL Cholesterol assay kit ab65390 
    - Cell-based Cholesterol assay kit ab133116 
    Cholesterol Efflux assay kit ab196985 
    Cholesterol Uptake assay kit ab236212

    Review our Metabolism Assay Guide to learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.

  • Platform

    Microplate reader

Properties

Images

  • Assessment of free and total cholesterol and cholesteryl ester levels useing ab65359. Protein levels of geranylgeranyldiphosphate synthase were measured by ELISA. Absolute amounts of CoQ10 were measured by HPLC as described in Materials and Methods. Cholesterol levels were quantified using a Cholesterol/Cholesteryl Ester Detection Kit from Abcam according to instruction. n?=?6, means ±SEM. Two-way ANOVA with Tukey's post-hoc test. * denotes statistical comparison between vehicle control (V) and high dose (H) and # denotes statistical comparison between water and ethanol treated groups. * and **, indicate significant difference at p<0.05, 0.01 and 0.0001, respectively. OWV; obese water vehicle, OWH; obese water high dose, OEV; obese ethanol vehicle, OEH; obese ethanol high dose.

  • Total cholesterol (free and ester forms) were determined with a cholesterol quantification assay (fluorometric detection) (ab65359) and are shown relative to untreated control cells.
  • Cholesterol and cholesteryl ester were quantified colourimetrically in biological fluids (dilution range 1:10-1:100; 50 minutes  incubation) and measured in duplicates (+/- SD).

  • Detection of Cholesterol/Cholesteryl Ester Using Cholesterol Quantitation Kit (ab65359). Cholesterol/Cholesteryl Ester was quantified using the kit by colorimetric (A) and fluorometric (B) methods according to the kit instructions. Background from the control reaction (without cholesterol) has been subtracted from each value. Note: Fluorometric assay is over 10 fold more sensitive than colorimetric assay.

Protocols

References

This product has been referenced in:

  • Tong XK  et al. High cholesterol triggers white matter alterations and cognitive deficits in a mouse model of cerebrovascular disease: benefits of simvastatin. Cell Death Dis 10:89 (2019). Read more (PubMed: 30692517) »
  • Jo Y  et al. Schnyder corneal dystrophy-associated UBIAD1 inhibits ER-associated degradation of HMG CoA reductase in mice. Elife 8:N/A (2019). Read more (PubMed: 30785396) »
See all 46 Publications for this product

Customer reviews and Q&As

1-10 of 39 Abreviews or Q&A

Good

Excellent Excellent 5/5 (Ease of Use)
Abreviews
It is very easy to handle, but it is recommended to extract it by other methods because it can dissolve the plate when extracting the lipid layer of the cell.

Miss. Ji Yeon Lee

Verified customer

Submitted Mar 11 2019

THE EFFET OF ATORVASTATIN-INDUCED TYPE-2 DIEBETIS

Excellent Excellent 5/5 (Ease of Use)
Abreviews
isolate the mitochondrial from cells and measure the cholesterol with the kit

Abcam user community

Verified customer

Submitted Jan 31 2018

Answer

Because phenol red has a strong absorbance/fluorescence at the wavelengths of the probe; therefore, it will interfere with the assay. The interference however would be minimal if the volume of the sample per well is low <20ul.

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Answer

ab65359 will extract total lipids, a large portion of which is from the plasma membrane in a normal cell.

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Answer



Ich habe im Labor nachgefragt und habe die folgende Antwort erhalten:

"Ideally, cells/tissues should be frozen before organic lipid extraction. Alternately, they can be stored after evaporation but this can lead to lipids sticking to the walls of the tubes which might become difficult to completely solubilize later."

Es wird nicht empfohlen, die Proben während der Chloroform: Isopropanol Extraktion einzufrieren. Danach ist es aber möglich, wenn auch nicht unbedingt empfohlen.

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Answer


We do not suggest extracting triglyceride and cholesterol in an unique extraction step with two layers. We have not tested this with each kit and cannot guarantee the effectiveness. Triglyceride is simply solubilized in detergent-water and total lipids are extracted in the cholesterol kit. We recommend preparing samples separately for best results. Since triglyceride can be hydrophobic, some of it can be in the organic phase or at the interphase. This could result in some loss if you separate the organic and aqueous phases.

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Answer


This kit is sold based on number of tests. A ‘test’ simply refers to a single assay well. The number of wells that contain sample, control or standard will be each counted as a test. Therefore this kit will be enough to perform less than 100 samples as standards and duplicates do indeed need to be considered.

You can use 96 welled plate wells to perform this assay however, please note that these are not included with the kit.

The kit can detect up to 5 ug/well for the colorimetric assay and up to 0.5 ug/well for the fluorometric assay.

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Question
Answer

Triton can generate background and hence we recommend NP-40. If it is absolutely essential for you to use Triton, you can run a blank using the triton solution to see what the interference is like.

We do carry NP-40 as ab142227 (link below)

https://www.abcam.com/np-40-ab142227.html

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Question
Answer

Hydrogen peroxide release is proportional to the cholesterol esterification reaction. H2O2 reacts with the probe to generate color at a downstream step.

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Answer

Thank you for contacting us.

Yes, you would see a pellet and an organic phase which contains all the cholesterol. This organic phase may seem to have multiple layers, so you did right in collecting all. This should be a max of 200 µl. Since it contains a high conc of chloroform and isopropyl alcohol, which evaporate fast. I would assume it take a couple of hrs to dry it out at 50°C. You can increase this time based on what you see.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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1-10 of 39 Abreviews or Q&A

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