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    products/assay-kits/cholesterol-cholesteryl-ester-assay-kit-quantitation-ab65359.pdf

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Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (ab65359)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (2)Q&A (37)References (156)

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Functional Studies - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (ab65359)
  • Functional Studies - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (ab65359)
  • ab65359 Cholesterol/Cholesteryl Ester Quantitation Kit
  • Functional Studies - Cholesterol/Cholesteryl Ester Quantitation Kit (ab65359)

Key features and details

  • Assay type: Quantitative
  • Detection method: Colorimetric/Fluorometric
  • Platform: Microplate reader
  • Assay time: 1 hr
  • Sample type: Cell culture supernatant, Other biological fluids, Plasma, Serum, Tissue Extracts, Urine

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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

    This product is manufactured by BioVision, an Abcam company and was previously called K603 Total Cholesterol and Cholesteryl Ester Colorimetric/Fluorometric Assay Kit. K603-100 is the same size as the 100 test size of ab65359.

    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.

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components Identifier 100 tests 2000 tests
    Cholesterol Assay Buffer WM 1 x 25ml 20 x 25ml
    Cholesterol Esterase (lyophilized) Blue 1 vial 20 vials
    Cholesterol Probe (in DMSO, anhydrous) Red 1 x 200µl 20 x 200µl
    Cholesterol Standard (2 µg/µl ) Yellow 1 x 100µl 20 x 100µl
    Enzyme Mix (lyophilized) Green 1 vial 20 vials
  • Research areas

    • Cardiovascular
    • Lipids / Lipoproteins
    • Lipid Metabolism
    • Cholesterol Metabolism
    • Signal Transduction
    • Metabolism
    • Lipid metabolism
    • Cardiovascular
    • Atherosclerosis
    • Lipoprotein metabolism
    • Kits/ Lysates/ Other
    • Kits
    • Cell Metabolism Kits
    • Lipid Metabolism Kits
    • Cholesterol
    • Kits/ Lysates/ Other
    • Kits
    • Cell Metabolism Kits
    • Lipid Metabolism Kits
    • Obesity
    • Kits/ Lysates/ Other
    • Kits
    • Cell Metabolism Kits
    • Lipid Metabolism Kits
    • Lipid metabolism
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Lipid and lipoprotein metabolism
    • Cholesterol Metabolism
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Lipid and lipoprotein metabolism
    • Lipoprotein metabolism
  • Relevance

    Cholesterol is an essential molecule in all animal life. It is involved in both normal development and diseases. A high level of serum cholesterol is an indicator for diseases such as heart disease. Cholesteryl ester is an ester of cholesterol. The ester bond is formed between the carboxylate group of a fatty acid and the hydroxyl group of cholesterol.

Associated products

  • Related Products

    • Cholesterol/Cholesteryl Ester Assay Kit - Detection (ab102515)
    • Cholesterol Assay Kit (Cell-Based) (ab133116)

Images

  • Functional Studies - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (ab65359)
    Functional Studies - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (ab65359)Mailloux RJ, Florian M, Chen Q, Yan J, Petrov I, Coughlan MC, et al., PLoS ONE 9(9): e106832., Fig 3, doi.org/10.1371/journal.pone.0106832

    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.

  • Functional Studies - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (ab65359)
    Functional Studies - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (ab65359)Singh, Natesh and Gerhard F Ecker., International journal of molecular sciences vol. 19,5 1278., Fig 1, doi:10.3390/ijms19051278
    Total cholesterol (free and ester forms) were determined with a cholesterol quantification assay (fluorometric detection) (ab65359) and are shown relative to untreated control cells.
  • ab65359 Cholesterol/Cholesteryl Ester Quantitation Kit
    ab65359 Cholesterol/Cholesteryl Ester Quantitation Kit

    Cholesterol and cholesteryl ester were quantified colourimetrically in biological fluids (dilution range 1:10-1:100; 50 minutes  incubation) and measured in duplicates (+/- SD).

  • Functional Studies - Cholesterol/Cholesteryl Ester Quantitation Kit (ab65359)
    Functional Studies - Cholesterol/Cholesteryl Ester Quantitation Kit (ab65359)
    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

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (156)

Publishing research using ab65359? Please let us know so that we can cite the reference in this datasheet.

ab65359 has been referenced in 156 publications.

  • Zhu Y  et al. Ceramide-mediated gut dysbiosis enhances cholesterol esterification and promotes colorectal tumorigenesis in mice. JCI Insight 7:N/A (2022). PubMed: 34914638
  • Liu J  et al. Curcumin sensitizes response to cytarabine in acute myeloid leukemia by regulating intestinal microbiota. Cancer Chemother Pharmacol 89:243-253 (2022). PubMed: 35066694
  • Xu M  et al. The E3 ubiquitin-protein ligase Trim31 alleviates non-alcoholic fatty liver disease by targeting Rhbdf2 in mouse hepatocytes. Nat Commun 13:1052 (2022). PubMed: 35217669
  • Zou D  et al. LncRNA MEG3 up-regulates SIRT6 by ubiquitinating EZH2 and alleviates nonalcoholic fatty liver disease. Cell Death Discov 8:103 (2022). PubMed: 35256601
  • Wu C  et al. Cholesterol Promotes Colorectal Cancer Growth by Activating the PI3K/AKT Pathway. J Oncol 2022:1515416 (2022). PubMed: 35528239
View all Publications for this product

Customer reviews and Q&As

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1-10 of 39 Abreviews or Q&A

Good

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

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
Abreviews
isolate the mitochondrial from cells and measure the cholesterol with the kit
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Jan 31 2018

Question

I want to analyse the conditioned media for cholesterol. Reading various papers, some suggest that the phenol red in cell culture media can affect the readings, can you comment if this is the case or would I be ok to try with the phenol media?

Read More

Abcam community

Verified customer

Asked on Jan 17 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.

Read More

Abcam Scientific Support

Answered on Jan 17 2018

Question

When extracting cholesterol from the liver tissues, will the chloroform: Isopropanol : NP-40 (7:11:0.1) system only extract intracellular cholesterol or it will also extract membrane lipids?

Read More

Abcam community

Verified customer

Asked on Nov 17 2014

Answer

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

Read More

Kevin Hanson

Abcam Scientific Support

Answered on Nov 17 2014

Question

At which point can samples be frozen within the sample prepaparation step?

Customer would like to freeze samples before the evaporation step after the chloroform: Isopropanol extraction.

Read More

Abcam community

Verified customer

Asked on Nov 13 2014

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.

Read More

Anja Hoffmann

Abcam Scientific Support

Answered on Nov 13 2014

Question

I just bought two kit to measure respectively cholesterol and triglyceride (ab65359 and ab65336)
I saw that the protocol for the cholesterol extraction suggests to use chloroform:isopropanol:NP-40 (7:11:0.1) while the protocol for the triglyceride suggests to use 5% NP-40 in H2O.
Is it possible to use one buffer for both so I do the extraction once and i save material?

Read More

Abcam community

Verified customer

Asked on Sep 26 2014

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.

Read More

Guillaume Boucher

Abcam Scientific Support

Answered on Sep 26 2014

Question

I need to know what is the the range of sensitivity of the assays? and how many sample I can measure by this kit? what is the meaning of 100 test? means, 100 sample or less that 100 sample because I must consider standards and duplication of each sample. If so, kit would be enough for only one 96 well plate?

Read More

Abcam community

Verified customer

Asked on Jun 17 2014

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.

Read More

Elisa Thomas

Abcam Scientific Support

Answered on Jun 17 2014

Question

Can Triton-x-100 be used in place of NP40 with this kit?

Read More

Abcam community

Verified customer

Asked on May 16 2014

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

Read More

Kevin Hanson

Abcam Scientific Support

Answered on May 16 2014

Question

Does probe react with H2O2 or the oxidized cholesterol?

Read More

Abcam community

Verified customer

Asked on Jul 23 2013

Answer

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

Read More

Kevin Hanson

Abcam Scientific Support

Answered on Jul 23 2013

Question

Hi I have a question about the following protocol for use with your
cholesterol ester quantation kit AB65359.
For cell or tissue samples:10^6 cells or 10 mg tissue can be extracted
with 200μl of chloroform: Isopropanol : NP-40(7:11:0.1) in a
micro-homogenizer. Spin the extract 5-10 minutes at 15,000 x g in a
centrifuge. Transfer all of the liquid (organic phase) avoiding the
pellet, to a new tube, air dry at 50°C to remove chloroform. Put the
samples under vacuum for 30 min to remove trace organic solvent.
Dissolve dried lipids with 200μl of Cholesterol Assay Buffer by
sonicating or vortexing until homogeneous (it is OK if the solution
becomes cloudy).
I used this protocol for adipose tissue and after the first 15K x g
spin I had several layers (not just one clear organic phase) I&apos;m
assuming some of this was lipids? However I&apos;m not sure which layer to
collect? When I attempted to collect all of the layers the solvent never
dried.
˜ How long should the air dry to remove chloroform step take? Just so I
know that I am on the right path.

Read More

Abcam community

Verified customer

Asked on Jun 13 2013

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.

Read More

Abcam Scientific Support

Answered on Jun 13 2013

1-10 of 39 Abreviews or Q&A

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