Coomassie Blue as a Near-infrared Fluorescent Stain: A Systematic Comparison With Sypro Ruby for In-gel Protein Detection

Abstract

Gel electrophoresis is an accessible, widely applicable and mature protein resolving technology. As the original top-down approach to proteomic analyses, among its many attributes the high resolution achievable by two dimensional gel-electrophoresis (2DE) 1 ensures that it remains an effective analytical technology despite the appearance of alternatives. However, in-gel detection remains a limiting factor for gel-based analyses; available technology generally permits the detection and quantification of only relatively abundant proteins (35). Many critical components in normal physiology and also disease may be several orders of magnitude less abundant and thus below the detection threshold of in-gel stains, or indeed most techniques. Pre-and post-fractionation technologies have been developed to address this central issue in proteomics but these are not without limitations (1-5). Thus improved detection methods for gel-based proteomics continue to be a high priority, and the literature is rich with different in-gel detection methods and innovative improvements (6 -34). This history of iterative refinement presents a wealth of choices when selecting a detection strategy for a gel-based proteomic analysis (35).Perhaps the best known in-gel detection method is the ubiquitous Coomassie Blue (CB) stain; CB has served as a gel stain and protein quantification reagent for over 40 years. Though affordable, robust, easy to use, and compatible with mass spectrometry (MS), CB staining is relatively insensitive. In traditional organic solvent formulations, CB detects ϳ 10 ng of protein in-gel, and some reports suggest poorer sensitivity (27,29,36,37). Sensitivity is hampered by relatively high background staining because of nonspecific retention of dye within the gel matrix (32,36,38,39). The development of

High-selectivity cytology via lab-on-a-disc western blotting of individual cells

“…The sparse starting sample consisted of 24 cells per a 15 μL suspension. Due to a poor sensitivity of total protein staining (> 1 ng), 43, 44 separation resolutions are calculated by analyzing fluorescence peaks of STAT3, GAPDH, β-TUB, and GFP. Across 14 cells, the 1.5-mm long protein separation axis resolved all proteins with separation resolution exceeding baseline resolution (Fig.…”

Coomassie staining provides routine (sub)femtomole in‐gel detection of intact proteoforms: Expanding opportunities for genuine Top‐down Proteomics

“…Accurate assessments of the concentration and purity of protein standards are necessary for meaningful determinations of stain sensitivity and selectivity. Apparent impurity of commercial protein preparations has been seen to varying degrees , and noted in other investigations in which protein concentration and purity were assessed . Yet it remains a problem in the field that efforts to examine the content/purity of commercial protein standards (and detailing methods to do so) are rarely reported if carried out at all.…”

“…Gel‐based purity analysis was essentially as described . Based on UV‐absorption concentration estimates, each protein standard was serially diluted to yield 1–0.0625 mg/mL solutions in 1DE sample buffer for 10–0.625 μg protein loads.…”

Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis

“…These comprehensive collections of detailed method descriptions have been edited by experts in the field, including Link [47], Reinders and Sickmann [48], Cramer and Westermeier [49], Kurien and Scofield [50], and Marengo and Robotti [51]. In-gel staining methods and routinely used detection technologies for studying 2D protein spot patterns have also been extensively described and critically examined in numerous publications [52,53,54,55,56,57,58,59,60]. The rapidly moving field of MS methodology analyzing peptides obtained from the proteolytic digestion of proteins is the subject of many excellent articles that outline in detail the many instruments and approaches available in modern proteomics research [61,62,63,64].…”