Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (ab65359)

Because phenol red has a strong absorbance/fluorescence at the wavelengths of the probe; therefore, it will interfere with the assay. The interference however would be minimal if the volume of the sample per well is low <20ul.

ab65359 will extract total lipids, a large portion of which is from the plasma membrane in a normal cell.

Ich habe im Labor nachgefragt und habe die folgende Antwort erhalten:

"Ideally, cells/tissues should be frozen before organic lipid extraction. Alternately, they can be stored after evaporation but this can lead to lipids sticking to the walls of the tubes which might become difficult to completely solubilize later."

Es wird nicht empfohlen, die Proben während der Chloroform: Isopropanol Extraktion einzufrieren. Danach ist es aber möglich, wenn auch nicht unbedingt empfohlen.

We do not suggest extracting triglyceride and cholesterol in an unique extraction step with two layers. We have not tested this with each kit and cannot guarantee the effectiveness. Triglyceride is simply solubilized in detergent-water and total lipids are extracted in the cholesterol kit. We recommend preparing samples separately for best results. Since triglyceride can be hydrophobic, some of it can be in the organic phase or at the interphase. This could result in some loss if you separate the organic and aqueous phases.

This kit is sold based on number of tests. A ‘test’ simply refers to a single assay well. The number of wells that contain sample, control or standard will be each counted as a test. Therefore this kit will be enough to perform less than 100 samples as standards and duplicates do indeed need to be considered.

You can use 96 welled plate wells to perform this assay however, please note that these are not included with the kit.

The kit can detect up to 5 ug/well for the colorimetric assay and up to 0.5 ug/well for the fluorometric assay.

Triton can generate background and hence we recommend NP-40. If it is absolutely essential for you to use Triton, you can run a blank using the triton solution to see what the interference is like.

We do carry NP-40 as ab142227 (link below)

https://www.abcam.com/np-40-ab142227.html

Hydrogen peroxide release is proportional to the cholesterol esterification reaction. H2O2 reacts with the probe to generate color at a downstream step.

Thank you for contacting us.

Yes, you would see a pellet and an organic phase which contains all the cholesterol. This organic phase may seem to have multiple layers, so you did right in collecting all. This should be a max of 200 µl. Since it contains a high conc of chloroform and isopropyl alcohol, which evaporate fast. I would assume it take a couple of hrs to dry it out at 50°C. You can increase this time based on what you see.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.