Overview

  • Product name
    Cholesterol Assay Kit - HDL and LDL/VLDL
    See all HDL LDL/VLDL kits
  • Detection method
    Colorimetric/Fluorometric
  • Sample type
    Urine, Serum, Plasma, Other biological fluids, Tissue Lysate
  • Assay type
    Quantitative
  • Assay time
    1h 00m
  • Product overview

    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.


    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

  • Notes

    If you need to purchase additional HDL precipitation buffer, please see ab105138.

    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

  • 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

  • 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 measure 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).

  • 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.

Protocols

References

This product has been referenced in:
  • Chae Y  et al. Trophic transfer and individual impact of nano-sized polystyrene in a four-species freshwater food chain. Sci Rep 8:284 (2018). Read more (PubMed: 29321604) »
  • Fernandes GW  et al. The Foxo1-Inducible Transcriptional Repressor Zfp125 Causes Hepatic Steatosis and Hypercholesterolemia. Cell Rep 22:523-534 (2018). Read more (PubMed: 29320745) »
See all 56 Publications for this product

Customer reviews and Q&As

1-10 of 60 Abreviews or Q&A

Total cholesterol from murine plasma samples

Average Average 3/5 (Ease of Use)
Abreviews
This kit offers the possibility of measuring total cholesterol or HDL and LDL/VLDL fractions separately, which is an advantage over having to purchase two different kits.
I used this kit to measure total cholesterol from murine plasma samples obtained by cardiac puncture and centrifugation (4 C, 20 min, 2000 g) diluted 1:10 with PBS. The kit was easy to use and precise when the results were compared with historical records.
However, when I tried the separation step for HDL and LDL/VLDL fractions, I had the feeling that the design of the kit is not perfect as it is very easy to disturb the pellet and get contamination of your HDL fraction.

Abcam user community

Verified customer

Submitted Sep 07 2018

I tried many different product to get HDL and LDL/VLDL better and more clear but failed then I bought Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390)to HDL and LDL/VLDL in obese mice serum to detect the effects of obesity on HDL and LDL/VLDL, as recommended in their datasheets and I never had any problem with it. I think its great product to use. I think this is best product to detect HDL and LDL/VLDL during in vivo studies

Abcam user community

Verified customer

Submitted Aug 09 2018

Measuring Cholesterol in Zebrafish Livers

Excellent Good 4/5 (Ease of Use)
Abreviews
I used the HDL and LDL/VLDL Cholesterol Assay Kit (Fluorometric) in order to measure total and free cholesterol in zebrafish liver. Overall the assay was successful. I did have some samples outside of the standard curve range meaning I will need to dilute my samples next time I run the assay. I divided all of my standard and sample fluorescence values by a factor of 10,000 prior to developing my standard curve. Attached is an image of my standard curve. From my standard curve I calculated the concentration of cholesterol that I then applied to the cholesterol concentration equation provided in the protocol. Some example data from my control sample is shown below.


Total Cholesterol: 57.7 ug/uL
Total HDL: 60.9 ug/uL
Total LDL/VLDL: 8.14 ug/uL

Free Cholesterol: 53.7 ug/uL
Free HDL :57.3 ug/uL
Free LDL/VLDL: 6.57 ug/uL

Cholesterol Esters: 3.99 ug/uL

Abcam user community

Verified customer

Submitted Dec 13 2017

Comparison of mouse liver extraction methods

Good Excellent 5/5 (Ease of Use)
Abreviews
I tried 2 extraction methods for mouse liver samples (from ApoE KO mice fed a high fat diet) that had been snap frozen and stored in -80 freezer.

I followed the Abcam sample preparation for tissue lysates and I also tried a protocol that had been posted by a previous reviewer of the product. See details below (copy pasted from previous reviewer):

For the separation of HDL and LDL/VLDL
1. Use 0.5% NP-40 in water to homogenize the tissue and then combine with the 2X precipitations buffer.
2. Mix 100 µL of sample with 100 µL of 2X Precipitation Buffer (1:1) in microcentrifuge tubes.
3. Incubate 10 minutes at room temperature.
4. Centrifuge at 2,000 x g (5,000 rpm on a bench-top microcentrifuge) for 10 minutes.
5. Transfer the supernatant into new labeled tubes. This is the HDL fraction.
6. Precipitates contain the LDL/VLDL fraction. To measure the LDL/VLDL fraction, centrifuge the precipitate again at 2,000 x g (5,000 rpm on a bench-top microcentrifuge at RT) for 10 minutes and remove trace amount of HDL supernatant carefully. I tissue homogenate you cannot measure the LDL fraction, only HDL!!!! (In tissue culture it is possibale).
7. Resuspend the precipitate in 200 µL PBS. This is the LDL/VLDL fraction.

If the supernatant is cloudy, the sample should be re-centrifuged. If the sample remains cloudy, dilute the sample 1:1 with PBS and repeat the separation procedure from step 2.

To homogenise the tissue I used a precellys 24 homogeniser.

Overall the protocols were similar but I preferred the Abcam one as all the reagents required are supplied within the kit. The alternative method requires NP-40 which is not supplied which would be expected.

From the image uploaded you can see that the 2 methods (Method 1 - Abcam Method 2 - user) produced very similar absorbance values (colorimetic method was used). The samples were diluted 5x to ensure they were within the standard curve.

Having compared the 2 methods I will proceed with using the Abcam method.

Overall this is a straightforward easy to use kit.

Abcam user community

Verified customer

Submitted Aug 14 2017

Abreviews
The kit is not sensitive enough to detect HDL-cholesterol in Huh-7 culture supernatant neither using colorimetric or fluorometric method

Abcam user community

Verified customer

Submitted Jul 07 2017

Abreviews
TISSUE PREPARATION
For total cholesterol
1. Homogenize 10 mg tissue in 200 μl of chloroform:Isopropanol:NP-40 (7:11:0.1) using a micro-homogenizer.
2. Spin the extract 10 minutes at 15,000 x g at RT in a centrifuge.
3. Transfer all of the liquid (organic phase) avoiding the pellet, to a new tube. Air dry the sup at 50°C to remove chloroform for 7min (in a hood).
4. Dissolve dried sup with Cholesterol Assay Buffer (1:1) by vortexing until homogeneous (it is OK if the solution becomes cloudy).

Note: The extraction procedure can be scaled up if larger amounts of sample are desired.

For the separation of HDL and LDL/VLDL
1. Use 0.5% NP-40 in water to homogenize the tissue and then combine with the 2X precipitations buffer.
2. Mix 100 µL of sample with 100 µL of 2X Precipitation Buffer (1:1) in microcentrifuge tubes.
3. Incubate 10 minutes at room temperature.
4. Centrifuge at 2,000 x g (5,000 rpm on a bench-top microcentrifuge) for 10 minutes.
5. Transfer the supernatant into new labeled tubes. This is the HDL fraction.
6. Precipitates contain the LDL/VLDL fraction. To measure the LDL/VLDL fraction, centrifuge the precipitate again at 2,000 x g (5,000 rpm on a bench-top microcentrifuge at RT) for 10 minutes and remove trace amount of HDL supernatant carefully. I tissue homogenate you cannot measure the LDL fraction, only HDL!!!! (In tissue culture it is possibale).
7. Resuspend the precipitate in 200 µL PBS. This is the LDL/VLDL fraction.

If the supernatant is cloudy, the sample should be re-centrifuged. If the sample remains cloudy, dilute the sample 1:1 with PBS and repeat the separation procedure from step 2.

Abcam user community

Verified customer

Submitted Mar 29 2016

Abcam HDL/VDL/Total cholesterol

Excellent Excellent 5/5 (Ease of Use)
Abreviews
I had no problems using this kit, it worked good on extracted lipids from liver tissue and values comparable to plasma levels.
Main limitation is the small amount of cholesterol assay buffer provided which just about fitted 100 samples. But we were aware of this and planned accordingly.
We extracted lipids from liver using one of the FAQ questions on the Abcam website related to ab65390 and that seemed to work.
All in all the experiment went good, we opted to assay total cholesterol and HDL only in plasma and liver samples which were 6 months old.

Abcam user community

Verified customer

Submitted Jul 09 2015

Answer


I can confirm that this kit is not antibody-based. A chemical polymer is used.

We aim to provide as much information as possible to customers, however I am sorry that on this occasion the exact details are proprietary.

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Question
Answer

I have contacted the lab regarding your question for ab65390.

After mixing with the Precipitation buffer and centrifuging, there should indeed be a pellet which would be the LDL fraction. The LDL fraction could be sticking to the walls of the tube and might need to look carefully against light to make sure you don’t miss it (especially if there is a small pellet).

You also need to make sure to convert the 2000g to rpm correctly for your centrifuge to ensure pellet formation. If sample volume is more than 200 ul total, spinning time can be increased to make sure LDL is precipitated.

Read More

Question
Answer


ab65390 HDL and LDL/VLDL Cholesterol Assay Kit can detect 1 ug and 0.1 ug cholesterol by the colorimetric and fluorometric versions respectively. Inter-assay variability from in-house QC results are within 5-10 %. Intra-assay variability is ˜2-3 %.

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1-10 of 60 Abreviews or Q&A

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