I need to calculate the concentration of Total cholesterol in rat tissue samples. can anyone provide me a colorimetric assay without using any kits
You can make the assay from the same components that are in the kit, if you can buy them. It's cheaper in the long run if you are going to do a lot of assays over an extended period of time, but you will have to invest some time to figure out how much of each ingredient to add.
This kit, for example, contains 3 enzymes, two substrates, a standard, and an assay buffer.
https://www.cellbiolabs.com/sites/default/files/STA-384-total-cholesterol-colorimetric-assay-kit.pdf
Needless to say, the manufacturers don't tell you what everything is or at what concentrations they are used. (The colorimetric probe is probably resorufin.)
2/21/20
Dear Kasturi,
I'm not sure what you are looking for. If you want to use an enzymatic method for cholesterol but don't want to purchase a kit, then you probably should follow Adam Shapiro's suggestion. It is a good one. His approach will save you some money, plus the enzymatic method has the advantage of specificity and reduced interference by other components in your sample matrix.
If you don't want to use an enzymatic method but prefer instead to use a simple colorimetric method, then you could use one of the older techniques. These are based on the Lieberman-Burchard detection system (See the attached files.) which has been around for many years. These work, although they do have some limitations as described in the attachments. They use inexpensive reagents commonly found in the lab, although some of the are harsh/hazardous.
I hope this information helps you. Good luck with your work.
Bill Colonna Dept. Food Science & Human Nutrition, Iowa State University, Ames, Iowa, USA [email protected]
Adam B Shapiro W.J. Colonna Thanks a lot for your inputs. Really appreciate it
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