I am trying to extract total protein from Rosellinia necatrix.
The purpose of extraction is to conduct SDS-PAGE with total protein followed by Western Blotting using anti-HA and anti-3XFLAG antibodies to detect a specific protein. I used several protocols (TCA precipitation, acetone precipitation, phenol methods) but found the protein concentration very low. It seems I need some better buffer for breaking open the cells. Please suggest me if you have any suitable protein extraction buffer composition/extraction methods for filamentous ascomycetes.
You do not say, how you break the cells. What are the methods you used?
How do you crush the frozen mycelia? Perhaps you have to do it more thoroughly. Is this a fungus with individual cell walls or is the intracellular content in a kind of communal tube?
I understand that you use liquid N2, but what tools or what procedure do you use to do the crushing?
Heating the mycelium in a solution of 3N HCl in acetone in a boiling water bath for 5 min completely dissolves the mycelium, leaving a clear solution. Proteins are not attacked in this procedure. However, the acidified acetone solvent may interfere with some protein assays (i.e. Lowry assay) . This interference may be avoided by aerating the sample to dryness.
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