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.

  • 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

  • Relevance
    HDL (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.
  • Alternative names
    • High density lipoprotein
    • LDL
    • Low density lipoprotein
    • Very low density lipoprotein
    • VLDL
    see all

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.

Cholesterol Assay Kit - HDL and LDL/VLDL images

  • 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.
  • 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

Protocols

References for Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390)

This product has been referenced in:
  • Ikhlef S  et al. Human paraoxonase 1 overexpression in mice stimulates HDL cholesterol efflux and reverse cholesterol transport. PLoS One 12:e0173385 (2017). Read more (PubMed: 28278274) »
  • Wang Y  et al. Renal Denervation Promotes Atherosclerosis in Hypertensive Apolipoprotein E-Deficient Mice Infused with Angiotensin II. Front Physiol 8:215 (2017). Mouse . Read more (PubMed: 28450836) »

See all 27 Publications for this product

Product Wall

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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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
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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.
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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.
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Abcam user community

Verified customer

Submitted Jul 09 2015


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.

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 ...

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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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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.



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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EDTA is not ideal for measurement of HDL/LDL in samples. EDTA plasma has potential disadvantages that have discouraged its use. Inadequate mixing can result in microclots. Also, EDTA osmotically draws water from red cells, diluting the plasma constitue...

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