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A modified and sensitive protein assay for dilute biological samples.

Cecular and Molecular

Thursday, February 23, 2012 Poster Session

Rajiv Heda (^), Upasana Kunwar *, Dominique Robinson *, Ghanshyam D. Heda *

* Department of Sciences and Mathematics, Mississippi University for Women, Columbus, MS

(^) Mississippi School for Mathematics and Science, Columbus, MS

There are a variety of protein assays that can measure the protein concentrations in biological samples at microgram levels. However, protein assays measuring the nanogram quantities in dilute biological samples is a rarity, except the recent surge in use of nanoparticles in the measurement. In this study, we aimed to improve and modify a protein assay that was originally described by Schaffner and Weissmann (Anal Biochem, 56, p 502-514, 1973). Our newly developed protein assay is rapid, reproducible, cost effective, and more importantly sensitive. Dilute protein samples of various concentrations were applied to the nitrocellulose membrane using a dot-blot apparatus and TCA precipitated. Precipitated protein spots were stained either with amido black alone or in combination with other protein stains. The combination staining of protein spots with amido black and colloidal gold (BioRad Laboratories) produced a synergistic effect, allowing us to measure the protein concentrations in nanogram range. The linearity of assay was confirmed by salt-elution of protein spots and analyzing with NanoDrop 1000 spectrophotometer (Thermo Scientific) at OD630, and/or by photo-scanning the stained protein spots and obtaining the pixel counts using Quantity One[R] analysis software (BioRad Laboratories). The validity of this assay at the microgram measurement levels was confirmed with the commonly used Biorad Protein Assay that is based on the principles of Lowry et al (J Biol Chem, 193, p 265-275, 1951). This newly developed protein assay will be useful in measuring protein concentration in minute biological samples prior to their biochemical analysis such as in comparative proteomics. We will be employing this protein assay in measuring the concentrations of low abundance proteins in endocytic vesicles that are immunopurified from mammalian cells expressing wild type and mutant CFTR. A mutated CFTR is responsible for the genetic disease cystic fibrosis.

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Title Annotation:Errata Sheet
Publication:Journal of the Mississippi Academy of Sciences
Article Type:Correction notice
Date:Apr 1, 2012
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