Cinnamic acid 4-hydroxylase (C4H) is an enzyme that catalyzes the conversion of cinnamic acid into caffeic acid and p-coumaric acid. Widely distributed in the soluble fractions of higher plants, yeasts, and fungi, it serves as a key enzyme in the intracellular lignin biosynthesis pathway. Research on this enzyme can facilitate the exploration of the metabolic mechanisms underlying lignin deposition during the developmental processes of various biological cells, thereby providing a theoretical basis for reducing the content of stone cells in fruits and improving their quality.
The Cinnamic Acid 4-Hydroxylase (C4H) Extraction Reagent is primarily designed for lysing plant tissues and extracting C4H from samples. This reagent is intended for research use only and is not suitable for clinical diagnosis or any other applications.
Materials to Be Prepared by the User
1. Distilled water
2. Centrifuge tubes or test tubes, homogenizer or mortar and pestle, refrigerated centrifuge
Operating Procedures (For Reference Only)
1. Take the plant tissue, rinse it thoroughly, and cut it into small pieces.
2. Add the Cinnamic Acid 4-Hydroxylase (C4H) Extraction Reagent at a ratio of 1 g plant tissue: 4 ml reagent, then fully homogenize or grind the mixture under ice bath conditions.
3. Centrifuge at 10,000×g and 4°C for 15-20 minutes, and collect the supernatant.
4. Store the supernatant at -20°C for subsequent Cinnamic Acid 4-Hydroxylase (C4H) activity assays or other experimental applications.
Calculation
Yield of crude enzyme solution from samples (ml/g)=Volume of supernatant (ml)/Mass of sample (g)×100%
Precautions
1. The sample to be tested must not contain phosphatase inhibitors, and repeated freeze-thaw cycles should be avoided.
2. If the measured value of the sample exceeds the upper limit of the standard curve, dilute the sample with the extraction reagent and perform the assay again.
3. Please use the reagent as soon as possible after opening to prevent it from affecting the results of subsequent experiments.
4. For your safety and health, wear a lab coat and disposable gloves during the operation.
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