Pre-Staining of Bacterial Cell Proteins as A Research And Diagnostic Tool
Journal Title: The Indian Practitioner - Year 2015, Vol 68, Issue 3
Abstract
We hereby describe a novel method of electrophoretic analysis of bacterial cell proteins prestained with a new reactive dye Coomassie Brilliant blue R-250 (CBB). The proposed method was standardised by studying as many as 950 disc electrophoretic separations, 50 variables, 60 isolates of Salmonella typhi and two standard NCTC S.typhi control strains. Under the critical conditions the cell free extract (CFE) of Salmonella and the soluble dye (8.5 mg/1 ml), Tris-glycine buffer (1:10), pH 8.3 was mixed in equal proportions, conjugate warmed at 41oC for 2 hours to ensure a stable covalent binding. 100 microlitre of this conjugated CFE was analysed by Polyacrylamide gel disc electrophoresis (PADGE). The method when compared with the post electrophoretic staining by Amidoblack revealed that the prestained discs were intensely well defined with a transparent gel. The amidoblack stained gel even with prolonged destaining showed a residual dye retention making identification of the faint components difficult. Proteins eluted from CBB-prestained gels retained its immunoreactivity, immunogenecity, purity and conjugates of Salmonella cell protein (mol. Wt. 69000) produced high titre monospecific antisera in immunised rabbit.
Authors and Affiliations
K Kaustubh, Rutuja K Kandle, J Vilas
Keywords
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