Name: Cholesterol Assay Kit - HDL and LDL/VLDL
SKU: ab65390
Rating: 4 (10 reviews)

Cholesterol Assay Kit ab65390 is used to quantify total cholesterol, free cholesterol, and cholesterol esters, and includes a method to separate HDL and LDL/VLDL fractions. The assay has one 60 min incubation, with colorimetric (570 nm) or fluorometric (535/587 nm) readout.

Individual kit components also available for purchase with a minimum order of 20 units. Contact us to discuss your needs.

Images

Functional Studies - Cholesterol Assay Kit - HDL and LDL/VLDL (AB65390), expandable thumbnail

Publications

Key facts

Detection method: Colorimetric/Fluorometric
Sample types: Tissue Lysate, Plasma, Serum, Other biological fluids
Assay type: Quantitative
Assay time: 1h

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Target data

See full target information N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-3-(3-methyl-4-oxo-2-phenylchromen-8-yl)prop-2-enamide

What's included?

100 Test

Components

2X LDL/VLDL Precipitation Buffer

1 x 10 mL

Assay Buffer 2

1 x 25 mL

Cholesterol Esterase

1 x 1 Vial

Cholesterol Standard

1 x 100 µL

Enzyme Mix I

1 x 1 Vial

OxiRed™ Probe

1 x 0.2 mL

Key facts

Detection method: Colorimetric/Fluorometric
Sample types: Tissue Lysate, Plasma, Serum, Other biological fluids
Assay type: Quantitative
Assay time: 1h
Assay Platform: Microplate reader

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage conditions: -20°C
Appropriate long-term storage conditions: -20°C
Storage information: -20°C

Notes

Cholesterol Assay Kit ab65390 uses a simple method to quantify total cholesterol, free cholesterol, and cholesterol esters in mammalian samples. It also includes an easy method to separate HDL and LDL / VLDL cholesterol.

How the assay works
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 hydrogen peroxide. Hydrogen peroxide is then processed by an enzyme causing a reaction with a probe to generate red 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

- Use complete sample for cholesterol measurement; or to separate HDL and LDL/VLDL, mix sample with precipitation buffer, incubate for 10 min, centrifuge for 10 min at 2,000 g, keep HDL fraction supernatant, repeat centrifugation and resuspend LDL/VLDL fraction pellet.
- For assay, 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 37°C.
- Analyze with microplate reader.

Related Cholesterol assay products
Cholesterol Assay Kit - HDL and LDL/VLDL ab65390 is identical to Cholesterol/ Cholesteryl Ester Assay Kit Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation ab65359, except for the addition of a buffer and protocol for separation of HDL and LDL/VLDL fractions.

Getting the best performance from ab65390
Note: This kit is primarily designed for use with serum and plasma samples. It has been successfully used with liver samples (including in Chang et al. 2019, PMID: 30670059, Yu et al. 2018, PMID: 29867579 and Sadh et al. 2017, PMID: 28800633); sample preparation in liver (and other tissue) samples may need optimization and modification by the user for successful use.

How other researchers are using Cholesterol assay kit ab65390
This cholesterol assay kit has been used in publications in a variety of sample types, including:

- Human: serum¹
- Mouse: plasma and liver tissue², plasma³, serum⁴,¹
- Rat: liver tissue⁵, serum¹, plasma⁶
- Fish: Z. temminckii serum⁷

^{References: 1 - Lin C et al 2018; 2 - Change RC et al 2019; 3 - Hinder et al 2019, Havighorst A et al 2019, Guo X et al 2017, Wen et al 2018, Fernandes GW et al 2018, Ahnstedt H et al 2018, Dumas SN and Ntambi JM 2018; 4 - Liu J et al 2018, Cui XB et al 2019, Rohm et al 2018; 5 - Yu S et al 2018; 6 - Park JB 2017; 7 - Chae Y et al 2018}

Related and recommended products
Other cholesterol assay kits include:

- Cell-based Cholesterol assay kit Cholesterol Assay Kit (Cell-Based) ab133116
- Cholesterol Efflux assay kit Cholesterol Efflux Assay Kit (Cell-based) ab196985
- Cholesterol Uptake assay kit Cholesterol Uptake Assay Kit ab236212

Cholesterol assay kit ab65390 is often used with Triglyceride assay kit Triglyceride Assay Kit - Quantification ab65336, and Free Fatty Acid assay kit Free Fatty Acid Assay Kit - Quantification ab65341, to study lipid metabolism and its role in various metabolic diseases such as diabetes, obesity, liver diseases, and cardiovascular conditions.

Learn more about our tools for obesity research, including antibodies and ELISA kits to adipogenesis and lipid metabolism markers, tools for GLP-1 receptor agonist research, and enzymatic assays to key biochemicals and enzymes involved in obesity-related metabolism.

Other notes
This product is manufactured by BioVision, an Abcam company and was previously called K613 HDL and LDL/VLDL Quantification Colorimetric/Fluorometric Kit. K613-100 is the same size as the 100 test size of ab65390.

Our broad range of gold-standard reagents features highly specific recombinant antibodies and kits, validated across applications and species to ensure the precision and reproducibility your work requires. Drive your drug discovery efforts forward with our tailored solutions designed to meet the unique challenges of obesity research.

REACH authorisation
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.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

HDL (High-Density Lipoprotein) and LDL (Low-Density Lipoprotein) often referenced together with VLDL (Very Low-Density Lipoprotein) are essential components in the regulation of cholesterol within the body. HDL known as "good" cholesterol is a spherical particle around 8-11 nm in diameter playing an important role in reverse cholesterol transport. LDL termed "bad" cholesterol and VLDL are involved in delivering cholesterol to cells. These lipoproteins are primarily expressed in the liver and the bloodstream. The LDL length varies from 18-25 nm while VLDL is larger around 30-80 nm. Their importance in cellular cholesterol homeostasis makes them frequent topics of study in lipid metabolism.

Biological function summary

HDL particles help remove excess cholesterol from peripheral tissues and transport it to the liver for excretion a process important for cardiovascular health. HDL particles are part of a multi-protein complex involving apolipoproteins such as ApoA-I ApoC and ApoE. LDL transports cholesterol to peripheral tissues but when accumulated it can lead to plaque formation in arteries. VLDL primarily acts as a triglyceride carrier converting into LDL after lipid delivery. These lipoproteins function synergistically forming a delicate balance essential for maintaining lipid levels.

Pathways

HDL and LDL are integral to cholesterol metabolism and energy homeostasis pathways. The reverse cholesterol transport pathway involves HDL in removing cholesterol from macrophages contributing to atheroprotection. LDL is linked to the endocytotic pathway associated with cellular uptake of cholesterol. APOLB is significant in the context of LDL production as it serves as the primary apolipoprotein in VLDL and LDL particles. Interaction between these pathways and lipoproteins determines systemic cholesterol levels and impacts atherosclerotic processes.

Associated diseases and disorders

Aberrations in HDL and LDL levels are closely associated with cardiovascular diseases like atherosclerosis and hyperlipidemia. An imbalance characterized by high LDL levels or low HDL contributes to plaque buildup in arterial walls leading to heart diseases. HDL and LDL are often modulated in therapeutic strategies aimed at improving cardiovascular health. The PCSK9 protein also plays a vital role where its inhibition leads to increased LDL receptor recycling and decreased blood LDL levels providing a therapeutic target in managing dyslipidemia and related disorders.

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8 product images

Functional Studies - Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390)
Rat serum samples were processed according to the protocol. The quantity of total (dilution range 1:10-1:100), HDL (dilution range 1:1-1:10), LDL/VLDL (dilution range 1:1-1:10) and free cholesterol (neat) was measured colourimetrically, in duplicates, after 40 minutes.
Li G et al., PLoS One, 7, e35895, 2012 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
Functional Studies - Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390)
Wild type (WT) or farnesoid X receptor knock-out (FRX-KO) mice were treated with either vehicle (Veh) or a high fat-containing diet (HFD) for 16 weeks. Serum HDL (top) and LDL/VLDL cholesterol (bottom) from 5-7 mice/group was measured using ab65390 following protocol instructions. An asterisk (*) means P<0.05 between WT and FXR-KO vehicle group. A pound (#) indicates P<0.05 between Wt and FXR-KO-HFD group. Image obtained from Li G et al., PLoS One, 2012; 7(4): e35895
Functional Studies - Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390)
Measurement of total cholesterol, HDL, LDL/VLDL from serum samples. Total Cholesterol (blue), HDL (green), and LDL/VLDL (cream) cholesterol were measured following the kit protocol.
Functional Studies - Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390)
Signal from standard curve was measured colourimetrically over a period of time. Background signal subtracted and each point on the curve represents duplicate values (+/- SD).
Biochemical assay - Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390)
Diagram showing the principles of the Cholesterol assay method.
Biochemical assay - Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390)
Triglyceride assay kit Triglyceride Assay Kit - Quantification ab65336, Cholesterol assay kit ab65390, and Free Fatty Acid assay kit Free Fatty Acid Assay Kit - Quantification ab65341 used with rat serum and rat liver.
Li et al. used Triglyceride assay kit Triglyceride Assay Kit - Quantification ab65336, Cholesterol assay kit Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation ab65359, and Free Fatty Acid assay kit Free Fatty Acid Assay Kit - Quantification ab65341 to investigate the impact of using L. reuteri treatment to ameliorate the impact of biorhythm disorder-ignited dyslipidemia in long-term darkness treated rats.
The concentrations of triglycerides (TG), total cholesterol (CHOL), HDL-cholesterol (HDL-C), and free fatty acids (NEFA) in rat sera and liver tissues were detected using Triglyceride Quantification Assay Kit (Triglyceride Assay Kit - Quantification ab65336), HDL and LDL/VLDL Cholesterol Assay Kit (ab65390). and Free Fatty Acid Quantification Assay Kit (Free Fatty Acid Assay Kit - Quantification ab65341), respectively. All procedures were carried out following the recommended instructions provided by the kit manufacturers.
Biochemical assay - Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390)
Cholesterol Assay Kit - HDL and LDL/VLDL ab65390 used with mouse serum.
Lee et al. used Cholesterol Assay Kit - HDL and LDL/VLDL ab65390 to investigate how thyme extract might mitigate high fat diet induced metabolic derangements and improve the gut environment.
Effects of thyme leaf extract (TLE) on serum indicators of liver function, lipid parameters, and blood glucose tolerance level in high-fat diet (HFD)-induced obese mice: Serum total cholesterol; serum HDL cholesterol; LDL cholesterol. ND, normal diet; HFD, high fat diet; PC, fructooligosaccharides fed + high fat; TLE50 and TLE100, thyme leaf extract high fat.
After a 12 h fasting period, mice were humanely euthanized by isoflurane exposure, and blood samples were collected. Serum was isolated by centrifuging the blood at 1500Ч g for 15 min and was stored at 80 °C. Biochemical analyses of the serum and liver samples were conducted using commercial ELISA kits: Abcam; Cholesterol, ab65390, Cambridge, UK.
Biochemical assay - Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390)
Triglyceride assay kit Triglyceride Assay Kit - Quantification ab65336 and cholesterol assay kit ab65390 used with mouse serum.
Zuo et al. used Triglyceride assay kit Triglyceride Assay Kit - Quantification ab65336 and Cholesterol assay kit ab65390 to investigate the impact of hepatocyte-specific AAV-mediated knockout of ANGPTL3 in mice.
Measurements of serum triglyceride (TG) and total cholesterol (TC) in mice at 1–4 weeks after AAV9-hAAT-Anc/Angptl3 administration. The control (Ctrl) mice received no treatments.
Blood samples collected from the animals were allowed for clotting at room temperature for 10 min. Serum was isolated by centrifugation at 5000 rpm for 10 min and stored at -80 °C in small aliquots. Determination of TG and TC was performed using the triglyceride and cholesterol assay kit (Abcam, Triglyceride Assay Kit - Quantification ab65336 and ab65390, respectively) according to the manufacture’s instruction.