Abstract
Quantitative proteome analyses suggest that the well-established stain colloidal Coomassie Blue, when used as an infrared dye, may provide sensitive, post-electrophoretic in-gel protein detection that can rival even Sypro Ruby. Considering the central role of two-dimensional gel electrophoresis in top-down proteomic analyses, a more cost effective alternative such as Coomassie Blue could prove an important tool in ongoing refinements of this important analytical technique. To date, no systematic characterization of Coomassie Blue infrared fluorescence detection relative to detection with SR has been reported. Here, seven commercial Coomassie stain reagents and seven stain formulations described in the literature were systematically compared. The selectivity, threshold sensitivity, inter-protein variability, and linear-dynamic range of Coomassie Blue infrared fluorescence detection were assessed in parallel with Sypro Ruby. Notably, several of the Coomassie stain formulations provided infrared fluorescence detection sensitivity to <1 ng of protein in-gel, slightly exceeding the performance of Sypro Ruby. The linear dynamic range of Coomassie Blue infrared fluorescence detection was found to significantly exceed that of Sypro Ruby. However, in two-dimensional gel analyses, because of a blunted fluorescence response, Sypro Ruby was able to detect a few additional protein spots, amounting to 0.6% of the detected proteome. Thus, although both detection methods have their advantages and disadvantages, differences between the two appear to be small. Coomassie Blue infrared fluorescence detection is thus a viable alternative for gel-based proteomics, offering detection comparable to Sypro Ruby, and more reliable quantitative assessments, but at a fraction of the cost.