Chromogen substrate TOPS determination of free fatty acid steps
The determination of free fatty acid (FFA) in serum is an important biochemical test index, and FFA is closely related to human health. Because serum components are complex and there are many types of FFA, the detection is affected by many factors, so the chromogen substrate TOPS is usually used to determine FFA.
Chromogen substrate TOPS reagent
Chromogen TOPS determination FFA steps:
1. In the container, first add the buffer solution weighed according to the formula ratio, and then add ATP, coenzyme A, 4-aminoantipyrine, ion activator (magnesium chloride), stabilizer (BSA), and preservative in sequence. After adding proper amount of water, add surfactant, adjust the pH to pH 6-9 with concentrated hydrochloric acid, add acyl-CoA synthase at last, then stir and filter, and add distilled water to the filtrate to obtain reagent A quantitatively.
2. Add buffer to the container, then add chromogen TOPS, water, and surfactant in sequence, adjust the pH to pH 6-9, finally add HRP and acetyl CoA oxidase, then stir and filter, and add distilled water to the resulting filtrate until the amount is reached Reagent B is obtained.
3. Fill the above reagents into vials and store them at 2-8°C. Take the reagent A and the sample and mix them in a certain ratio, incubate for a certain period of time, and read the absorbance value A1; add the reagent B and mix evenly, incubate for a certain period of time, and read the absorbance value A2. FFA concentration in the sample=standard solution concentration Χ(ΛΑ_/ΛΑ_#), ΛΑ_=Α2-A1.
In a clean glass container, first add HEPES buffer (Good's buffer, zwitterionic buffer, including HEPES, Tris, MES, MOPS, etc.) that has been weighed according to the formula ratio, and then add appropriate amount of water and then add 5ml of surfactant. L TritonX-100, use concentrated hydrochloric acid to adjust the pH to pH 6-9, the amount is about 5ml/L, then stir and filter, and then add distilled water to the obtained filtrate to a quantitative amount.
The method of using TOPS as a chromogen substrate to detect serum or plasma FFA has high accuracy and small measurement error, and it is the most suitable color among the many Trinder's chromogens, which is low in acetyl CoA, high in stability, and large in molar absorption coefficient. original. In addition to TOPS, the chromogen substrates produced by Desheng include TOOS, MAOS, ADPS, etc., which are suitable for the detection of blood sugar and other biochemical indicators.
HEPES, as a zwitterionic buffer, increases the osmotic pressure of the cell culture system by increasing the concentration of solution ions, maintaining normal cell morphology and function, and improving cell survival rate. Widely used in cell culture, especially under specific conditions such as tumor cell culture, it is crucial to maintain cell growth and function.