I am working on filamentus fungi, I culture it on broth culture and find the best time for extraction but still I cannot make sure that the way of extraction that I used is correct, actually I think that it is wrong. i need to extract the pectinase and get SDS-PAGE results from it.
Danita Pachideh
Read the method of Isolation and identification of pectinolytic microfungi conducted by Okafor et al 2010 in their good research paper (Pectinolytic activity of wild-type filamentous fungi fermented on agro-wastes ) . This may give you new ideas.
Hi Danita Pachideh Primarily you have to identify the cell wall constituents and then treat them with pertinent cell wall degrading enzymes. In addition, You may go for a sonication. But you should have a piece of background knowledge on the location of pectinases, whether in the cytoplasm or cell wall. Then you go for a specific cellular or extracellular extraction. Further, the cell-free extracts should be run in a column chromatography to get a protein fraction using relevant mobile phases. Then two-dimensional gel electrophoresis is recommended in comparison to SDS PAGE. Hope this will add some insights.
Hi Danita,
As mentioned above, you need to find out where it is. Whether you know if your extraction method is correct or not will depend on how much time you have to do different experiments with different methodologies. You have certainly read articles about it and maybe they vary a lot in the extraction methodology. Choose some and adapt them to what you have available in the laboratory (equipment, reagents ...) and try to find a good method for the fungus you work with and in which the pectinase yield is good. There are several steps to purify an enzyme, the right steps are discovered based on trial and error.
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