In-gel proteomics : optimisation of Coomassie Brilliant Blue staining as a fluorescent alternative for sensitive protein detection

Abstract

Gel electrophoresis, particularly one-dimensional electrophoresis (1DE) and two-dimensional electrophoresis (2DE), remain among the most widely used methods for resolving and analysing a variety of proteomes. Detection of the resulting proteome map relies on the stain employed (i.e. Coomassie Brilliant Blue (CBB) or SYPRO Ruby (SR) in addition to many others). Fluorescent in-gel protein stains are generally preferred due to their higher sensitivity, reduced background interference, and wider dynamic range. Although traditionally used densitometrically, CBB has been shown to possess fluorescent properties. Recently, it was noted that infrared detection of CBB stained proteins was similar to SR which suggested a competitive alternative for sensitive fluorescent staining. Systematic characterisation of numerous CBB formulations in the Coorssen Lab identified BioSafe (Bio-Rad) and the Neuhoff formulation (NG) as superior performers; however, application to native proteomes saw slightly poorer detection in comparison to SR. Sub-optimal performance of the CBB stains tested might well have been due to the standardised protocol applied at the time. Using protein standards and 1DE, the protocol for both BioSafe and NG were optimised to improve selectivity without affecting sensitivity; the resulting linear dynamic range for BioSafe and NG were similar to that of SR. Although the capacity for detection was investigated using purified proteins, the ultimate goal of this research program is the improved detection of native proteomes. 2DE analyses of mouse brain and A. thaliana (leaf) proteomes indicate superior total spot detection using CBB, in particular NG, relative to SR and BioSafe. Thus, although SR is widely employed for sensitive protein detection, the same or better analyses can be achieved using CBB, at a fraction of the cost.

Date of Award	2012
Original language	English