Preparing TOOS Solution for Chromogen Substrates with Hands - Ultra Complete Introduction
Release time:
2023-08-17
Chromogen substrates play a crucial role in biochemical research, as they can catalyze measurable signals with enzymes to reveal reaction processes. TOOS is such a widely used chromogen substrate in biochemical experiments, which is stable enough to be used in both solution and experimental pipeline detection systems, as well as in reagent kits to meet clinical requirements. How to prepare and test TOOS, which is mostly powdered in the market?
1、 Preparation of TOOS solution for chromogenic substrates
1. Dissolve 20 mg of TOOS in 10 ml PBS (phosphate buffer) to prepare a 6.6 mM TOOS solution. It is necessary to adjust the solubility according to the experimental needs to obtain the required concentration.
2. Dissolve 14 mg of 4-aminoantipyrine (4-AA) in 10 ml PBS to prepare a 6.6 mM 4-AA solution.
3. Prepare a 2 U/ml horseradish peroxidase solution using PBS as the solution substrate.
4. Mix the various solutions mentioned above by equal volume to prepare the detection solution. The prepared detection solution needs to be stored at 4 ℃ in a dark environment.
2、 Detection steps of chromogenic substrate TOOS
(1) Prepare sample solutions for enzymatic oxidation reactions, ensuring that the pH range of the buffer is between 5.5 and 9.5 to ensure optimal enzyme activity.
(2) Prepare a standard solution containing a known substrate concentration using the same buffer solution for preparing the standard curve.
(3) Add an appropriate amount of oxidase to the sample solution, and then add an equal volume of pre prepared detection solution.
(4) Incubate the mixture at room temperature or 37 ℃ for 30 minutes to 1 hour to ensure sufficient enzymatic reaction.
(5) After the reaction is completed, the optical density (O.D. value) at 555 nm is measured, which can reflect the formation of enzyme catalyzed products.
(6) Use the prepared standard solution to create a standard curve and determine the substrate concentration in the sample solution accordingly.
3、 Precautions
When preparing the detection solution, it is necessary to strictly mix according to the proportion required by the experiment to ensure the accuracy of the reaction. When storing the detection solution, it is important to avoid light at 4 ℃ to prevent the decomposition of the chromogenic substrate.
The preparation and detection steps of chromogenic substrate TOOS are relatively simple, but they play an important role in biochemical experiments. Through reasonable operating procedures, we can obtain reliable experimental results and provide strong support for biochemical research. As a professional manufacturer of chromogenic substrates, Desheng can provide a wide range of stable raw material substrates, including TOOS, TOPS, ADOS, ADPS, ALPS, DAOS, HDAOS, MADB, MAOS, etc. If you are interested, welcome to come and purchase!
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