Type of stains used in detection of protein in gel

Abstract

Protein separation by two-dimensional electrophoresis (2DE) is largely used in proteomic approaches because of both high resolution and the availability of powerful image analysis software for gel comparison and compatibility with subsequent protein characterization by mass spectrometry [1]. For these various aspects, the selection of the protein staining procedure is of major importance [2]. Based on two independent biochemical characteristics of proteins, 2DE combines isoelectric focusing, which separates proteins according to their isoelectric point, and SDS-PAGE, which separates them further according to their molecular mass . The next typical steps of the flow of gel-based proteomics are spots visualization and evaluation, expression analysis, and finally protein identification by mass spectrometry. In order to take advantage of the high resolution capacity of 2DE, proteins have to be completely denatured, disaggregated, reduced and solubilized to disrupt molecular interactions and to ensure that each spot represents an individual polypeptide. Proteins can be stained before the open access Materials (pre-electrophoretic protein stain), or after 2DE separation (post-electrophoretic protein stain). Classically, Coomassie blue was the most widely used non-covalent dye for post-electrophoretic protein staining [3]. However, it suffers from a low sensitivity in protein detection, including in the improved colloidal version [4]. In contrast, the other classical protein stain, silver nitrate, displays an excellent sensitivity but could interfere with protein analysis by mass spectrometry [5]. In the last decade, different fluorescent dyes have been introduced [6]. These encompass Sypro Ruby [7], and Ruthenium red-based dyes [8]. However, their present use remains relatively limited, probably due to their cost and/or technical difficulties.