Overview

  • Product name
    Cholesterol Assay Kit - HDL and LDL/VLDL
    See all HDL LDL/VLDL kits
  • Sample type
    Urine, Serum, Plasma, Other biological fluids, Tissue Lysate
  • Assay type
    Quantitative
  • Assay time
    1h 00m
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Human
    Predicted to work with: Mammal
  • Product overview

    Abcam's Cholesterol Assay Kit (ab65390) provides a simple quantification method of HDL and LDL/VLDL after a convenient separation of HDL from LDL and VLDL (very low-density lipoprotein) in serum samples. In this assay, cholesterol oxidase specifically recognizes free cholesterol and produces a component that will react with probe to generate color (570 nm) and fluorescence (Ex/Em = 538/587 nm). Cholesterol esterase hydrolizes cholesteryl ester into free cholesterol, therefore, cholesterol ester and free cholesterol can be detected separately in the presence and absence of cholesterol esterase in the reactions.


    Visit our FAQs page for tips and troubleshooting.


    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.

  • Notes

    Regulation of HDL (high-density-lipoprotein)-cholesterol and LDL (low-density-lipoprotein)-cholesterol plays a central role in various disease developments. It is well known that low levels of HDL and high level of LDL are associated with an increased risk of cardiovascular events.

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

  • Tested applications
    Suitable for: Functional Studiesmore details

Properties

Applications

Our Abpromise guarantee covers the use of ab65390 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Functional Studies Use at an assay dependent dilution.

Images

  • HDL and LDL/VLDL Cholesterol Assay Kit (ab65390)
    HDL and LDL/VLDL Cholesterol Assay Kit (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

  • ab65390 HDL and LDL/VLDL Cholesterol Assay Kit
    ab65390 HDL and LDL/VLDL Cholesterol Assay Kit

    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.

  • ab65390 HDL and LDL/VLDL Cholesterol Assay Kit
    ab65390 HDL and LDL/VLDL Cholesterol Assay Kit

    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).

  • Functional Studies - HDL and LDL/VLDL Cholesterol Assay Kit (ab65390)
    Functional Studies - HDL and LDL/VLDL Cholesterol Assay Kit (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.

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) »
  • Zimmer M  et al. CAT-2003: A novel sterol regulatory element-binding protein inhibitor that reduces steatohepatitis, plasma lipids, and atherosclerosis in apolipoprotein E*3-Leiden mice. Hepatol Commun 1:311-325 (2017). Read more (PubMed: 29404461) »

See all 40 Publications for this product

Publishing research using ab65390? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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58 reviews or Q&A

10
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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
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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.
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Submitted Aug 14 2017

Measure HDL cholesterol in supernatant of Huh-7 cells

 Poor Poor 1/5 (Ease of Use)
Abreviews
The kit is not sensitive enough to detect HDL-cholesterol in Huh-7 culture supernatant neither using colorimetric or fluorometric method
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Submitted Jul 07 2017

HDL and total cholesterol on liver tissue homogenates

 Good Good 4/5 (Ease of Use)
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.
abreview image
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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.
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Submitted Jul 09 2015

Hi AbCam
Just enquiring as to how this LDL/VLDL precipitation system works. Is it by dextran sulphate? Or an antibody?
Thanks


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.

Pellet formation for ab65390

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 ...

Read More

Hi, I am wondering, what are the LOD, Intra and Inter-assay variation for this kit?


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...

Read More


Recently we purchased HDL and LDL/VLDL cholesterol assay kit ab65390.
We do not have the OD 570 for microtiter plate reader.
Can we use other measurement? Such as 490 or 630nm.



Unfortunately, this kit can only be read on a colorimeter at an OD of 570 nm and on a fluorometer at Ex/Em = 538/587 nm.



I have another question regarding this kit and this time is about the standard curve. I have done so far 5 experiments and in neither of them the the standard curve is linear. I am attaching an excel file with all the standard curves i made so far. Of ...

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Thank you for your inquiry.

A couple of points to note:

- Please subtract the zero std from all std values before making the std curve.

- Our kit directions mention preparing std from 0. 0.1, 0.2, 0.3, 0.4 ...

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