May 14, 2019
UC Davis - Total Cholesterol (TC) Protocol
- Peter Havel1
- 1University of California, Davis
- Mouse Metabolic Phenotyping CentersTech. support email: info@mmpc.org
External link: https://mmpc.org/shared/document.aspx?id=92&docType=Protocol
Protocol Citation: Peter Havel 2019. UC Davis - Total Cholesterol (TC) Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.ygeftte
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 22, 2019
Last Modified: May 14, 2019
Protocol Integer ID: 20710
Keywords: Cholesterol
Abstract
Summary:
Cholesterol esters are enzymatically hydrolysed by cholesterol esterase to cholesterol and free fatty acids. Free cholesterol, including that originally present, is then oxidized by cholesterol oxidase to cholest-4-en-3one and hydrogen peroxide. The hydrogen peroxide combines with HBA and 4-aminoantipyrine to form a chromophore (quinoneimine dye) which may be quantitated at 500-550nm.
Materials
MATERIALS
CalibratorFisher DiagnosticsCatalog #TR43002
ReagentsFisher DiagnosticsCatalog #TR13421
PBS
Microplate
Platereader
Note:
Thermo Fisher Scientific, RRID:SCR_008452
Add 5 µl of calibrator and sample to each well.
Add 300 µl of reagent to each well. Incubate at 37°C for 5 minutes. Read at 540 nm.
Subtract blank readings from final readings. The assay will be linear so the unknown samples can be calculated as (Sample Absorbance ÷ Calibrator Absorbance) × Calibrator Concentration.