In this experiment, polyacrylamide gel electrophoresis was used to analyze and compare the peroxide isoenzymes, and their activities were determined by guaiacol method. (Source: Laboratory instruction of biochemistry and molecular biology, Shao Xueling et al, Wuhan University Press, 2003)

Operation method

polyacrylamide gel electrophoresis

Principle

Different species and even different varieties of organisms not only have variations in external morphology, but also have certain differences in their expression products, enzymes and proteins, due to differences in structural genes. Therefore, isozyme analysis, as a genetic analysis tool, has been widely used in various fields of agriculture and forestry, such as varietal resources survey, determination of hybrid progeny, seed purity identification, etc., and has played a great role in guiding production. In addition, isozyme analysis is also of positive significance to the study of the affinity relationship between organisms of different origins and genera, and to the development and utilization of medicinal plant resources.

Materials and Instruments

Guaiacol
Benzidine Ice Acetic Acid Sodium Acetate Manganese Sulfate Hydrogen Peroxide TMEM Ammonium Persulfate Agar
Electrophoresis apparatus Vertical plate electrophoresis tanks Microsampler Beakers Test tubes Long droppers Petri dishes

Move

1. Sample solution preparation: Take 1 g of the material stem with twice cold purified water, fully grind it into homogenate in a pre-cooled mortar and pestle in the refrigerator, then centrifuge (3500 r/ min, 20 min), add equal volume of glycerol and a little bromophenol blue indicator.

2. Gel preparation (according to "conventional polyacrylamide gel electrophoresis"), sample volume of 40 μl/well, voltage 180 V, electrophoresis time 2.5-3 h. The gel was prepared in the same way as that of the conventional polyacrylamide gel electrophoresis.

3. Staining: 0.5 g of benzidine was dissolved in 250 ml of 10% glacial acetic acid to form benzidine mother liquor. 1.35 g of guaiacol was dissolved in 250 ml of 10% glacial acetic acid to form mother liquor. The staining solution was 100 ml 0.2 mol/L sodium acetate solution, 10 ml 5 mmol/L manganese sulfate solution, 25 ml benzidine masterbatch, 40 ml guaiacol masterbatch, and 25 ml 0.12% hydrogen peroxide solution (prepared before use). The gel was washed out and transferred to the staining solution, placed at a constant temperature of 37 ℃ for 30 min, after color development, stored in 7% acetic acid and plotted or photographed.

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