Catalase (CAT), also known as contact enzyme, is a type of conjugate enzyme with iron porphyrin as the prosthetic group. It is a tetrameric enzyme composed of four identical subunits, containing a total of 4 molecules of ferrous heme as prosthetic groups, with a molecular weight of approximately 24 KD. CAT can rapidly scavenge hydrogen peroxide, a toxic substance produced by cellular metabolism, and work together with GSH-Px to protect sulfhydryl enzymes, membrane proteins and dissociate hydrogen peroxide.
The Plant Catalase (CAT) Extraction Reagent is primarily designed for lysing plant tissues and extracting catalase from plant 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 fresh plant tissues (or animal tissues) under normal or stress conditions, rinse them thoroughly, blot them dry, cut them into small pieces, and weigh them quickly.
2. Add pre-cooled Plant Catalase (CAT) Extraction Reagent at a ratio of 0.5 g plant tissue: 2 ml reagent, then fully homogenize or grind the mixture under ice bath conditions.
3. Transfer the homogenate into a 15 ml centrifuge tube, rinse the mortar or homogenizer with Plant Catalase (CAT) Extraction Reagent, combine the rinsing solution into the same centrifuge tube, and make up the volume to 10 ml with the reagent.
4. Mix well, let it stand in a 4°C refrigerator for 10 minutes, then transfer the upper clear solution into a new centrifuge tube.
5. Centrifuge at 1,200×g and 4°C for 30 minutes, collect the supernatant, which is the crude catalase extract.
6. Store the supernatant at 4°C for later use in CAT 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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