Detection of Plant Nematodes by Isozyme Analyses

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Isozymes, as a class of proteins, exist widely in organisms. The main separation methods include electrophoresis, chromatography, enzymatic method, and immunological method. Enzymatic phenotyping is one of the first non-morphological based methods used for nematode identification. After the soluble protein is extracted from the plant nematode, the specific isoenzymes are stained, and the different nematode species can be identified by polyacrylamide gel electrophoresis analysis.

Lifeasible makes full use of our specialized libraries, selection strategies, and maturation techniques to optimize the stability and performance of plant protection. With advanced technology and experienced staff, we provide protocols for the detection of plant nematodes to global clients to support their research.

Detection of Plant Nematodes by Isozyme Analyses

Specific isozymes on nematode proteins are stained and analyzed by polyacrylamide gel electrophoresis according to the molecular structure, activity, and immunogenicity of different isozymes, which show the differences in charge, molecular weight, and conformation, to identify different nematode species.
Lifeasible provides detection of plant nematodes with isozymes, such as malate dehydrogenase, cellulase, superoxide dismutase, esterase, and glutamate-oxaloacetate transaminase.

Fig.1 A polyacrylamide gel showing the esterase and malate dehydrogenase phenotypes of plant nematode species. (Esbenshade PR and Triantaphyllou AC., 1990)

Workflows of Isozyme Analyses

Steps	Operation Methods
Enzyme Solution Extraction of Plant Nematodes	Nematodes are separated by the funnel method, washed with distilled water, counted, and concentrated. The extract is added at 4°C, ground thoroughly in an ice bath, centrifuged for 15 min with 10000 g, and the supernatant is taken as an electrophoresis sample.
Making Glue	Pour the prepared separation glue into the glass plate immediately, and slowly pour the concentrated glue after the gel is polymerized. Then let the mold stand still for an hour, pour the electrode buffer into the electrophoresis tank, and prepare the sample.
Adding samples	8 μL Sample is added to each well, and then one drop of bromophenol blue solution is added to the upper tank.
Electrophoresis	We perform the vertical polar polyacrylamide gel electrophoresis. The electrophoresis tank is placed horizontally in the refrigerator, maintaining the temperature at 4°C to keep the enzyme's activity.
Staining	Staining esterase with fast blue R-α-naphthalene acetate method. Staining malate dehydrogenase and superoxide dismutase with the double color method. Staining cellulase with the iodine-potassium iodide method. Staining catalase with the VC-benzidine method. Staining soluble protein with the trichloroacetic acid-Coomassie brilliant blue method. After staining, the samples are immediately fixed with 10% acetic acid solution for more than 3 h and photographed.

Lifeasible offers reliable protocols for the detection of plant nematodes to meet your research demands. With years of experience in plant science, our advanced platforms can help our clients solve various difficulties and conduct research. If you are interested in our services or have any questions, please feel free to contact us or make an online inquiry.

Reference

Esbenshade PR and Triantaphyllou AC. (1990). "Isozyme phenotypes for the identification of Meloidogyne species." Nematol. 22, 10-15.

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