Canadian Forest Service Publications

An innovative approach to the determination of nanogram quantities of protein whereby the interference by sodium dodecyl sulfate (SDS) and mercaptoethanol was eliminated. 1992. Ekramoddoullah, A.K.M. FASEB (Federation of American Societies for Experimental Biology) Journal 6: 4&5.

Year: 1992

Issued by: Pacific Forestry Centre

Catalog ID: 3139

Language: English

Availability: Not available through the CFS (click for more information).

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Conifer foliage is rich in phenolic substances which interfered with the electrophoretic separation of foliar proteins. A method was therefore developed that allowed the extraction of protein amenable to separation by electrophoresis. We are now employing two-dimensional gel electrophoresis to analyze proteins of conifer foliage whose biosynthesis are affected by inoculation with a fungal pathogen. A meaningful evaluation of the electrophoretic data can be made if the samples to be analyzed are compared on an equal protein basis. However, SDS and mercaptoethanol used in the extraction and solubilization of proteins interfered with the available methods of protein determination. The interference was pronounced when samples contained small amounts of protein. In this study, a method employing membrane, laser-based scanning, and computer software technology was used for the quantitation of nanogram quantities of proteins. The method is sensitive enough to quantitate small amounts of proteins in the presence of SDS and mercaptoethanol. The recommended procedure involves spotting of the protein solution (1 microlitre) on a PVDF membrane, staining with Coomassie blue, destaining, scanning, detection and quantitation.