Key features and details
- Assay type: Quantitative
- Detection method: Colorimetric/Fluorometric
- Platform: Microplate reader
- Assay time: 1 hr
- Sample type: Other biological fluids, Plasma, Serum, Tissue Lysate, Urine
Product nameCholesterol Assay Kit - HDL and LDL/VLDL
See all HDL LDL/VLDL kits
Sample typeUrine, Serum, Plasma, Other biological fluids, Tissue Lysate
Assay time1h 00m
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.
In the cholesterol assay protocol, cholesterol oxidase acts on free cholesterol to produce a chemical which reacts with a probe to generate color (570 nm) and fluorescence (Ex/Em = 538/587 nm). Cholesterol esterase is used to hydrolyze cholesteryl ester into free cholesterol.
If cholesterol esterase is included in the assay, total cholesterol is measured. If it is not included, free cholesterol is measured. The amount of cholesterol ester can be calculated by subtracting free cholesterol from total cholesterol.
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, Yu et al. 2018, and Sadh et al. 2017); sample preparation in liver (and other tissue) samples may need optimization and modification by the user for successful use.
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 37ºC
- analyze with microplate reader
Other cholesterol assay kits include:
- Cell-based Cholesterol assay kit ab133116
- Cholesterol/Cholesterol Ester assay kit ab65359
- 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.
How other researchers have used 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
Storage instructionsStore at -20°C. Please refer to protocols.
Components Identifier 100 tests 2X LDL/VLDL Precipitation Buffer NW 1 x 10ml Cholesterol Assay Buffer WM 1 x 25ml Cholesterol Esterase (Lyophilized) Blue 1 vial Cholesterol Probe (in DMSO, anhydrous) Red 1 x 200µl Cholesterol Standard (2 ug/ul) Yellow 1 x 100µl Enzyme Mix (Lyophilized) Green 1 vial
- Pathways and Processes
- Metabolic signaling pathways
- Lipid and lipoprotein metabolism
- Cholesterol Metabolism
RelevanceHDL (high-density-lipoprotein), LDL (low-density lipoprotein) and VLDL (very low-density lipoprotein) are lipoproteins that act as carrier proteins for cholesterol. The regulation of HDL-cholesterol and LDL-cholesterol plays a central role in various disease development. It is well known that low levels of HDL and high level of LDL are associated with an increased risk of cardiovascular events.
- High density lipoprotein
- Low density lipoprotein
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
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.
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.
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).
ab65390 has been referenced in 98 publications.
- Boada C et al. Rapamycin-Loaded Biomimetic Nanoparticles Reverse Vascular Inflammation. Circ Res 126:25-37 (2020). PubMed: 31647755
- Li Q et al. C-Reactive Protein Causes Adult-Onset Obesity Through Chronic Inflammatory Mechanism. Front Cell Dev Biol 8:18 (2020). PubMed: 32154244
- Boujon V et al. Dual PPARa/? agonist aleglitazar confers stroke protection in a model of mild focal brain ischemia in mice. J Mol Med (Berl) 97:1127-1138 (2019). PubMed: 31147725
- Liebig M et al. Endogenously increased n-3 PUFA levels in fat-1 transgenic mice do not protect from non-alcoholic steatohepatitis. Hepatobiliary Surg Nutr 8:447-458 (2019). PubMed: 31673534
- Chang RC et al. Programmed increases in LXRa induced by paternal alcohol use enhance offspring metabolic adaptation to high-fat diet induced obesity. Mol Metab 30:161-172 (2019). PubMed: 31767168