Assessing Detection Methods for Gel-Based Proteomic Analyses

Abstract

Proteomic analyses using two-dimensional gel electrophoresis (2DE) depend heavily upon the quality of protein stains for sensitive detection. Indeed, detection rather than protein resolution is likely a current limiting factor in 2DE. The recent development of fluorescent protein stains has dramatically improved the sensitivity of in-gel protein detection and has enabled more accurate protein quantification. Here, we have evaluated the overall quality and relative cost of five commercially available fluorescent stains, Krypton, Deep Purple, Rubeo, Flamingo, and the most commonly used stain, Sypro Ruby (SR). All stains were found to be statistically comparable with regard to number of protein spots detected, but SR was superior with regard to fluorophore stability (e.g., capacity for repeated use of the stain solution). Notably, colloidal Coomassie Blue was also found to be comparable to SR when detected using an infrared fluorescence imaging system rather than standard densitometry. Thus, depending on available equipment and operating budgets, there are at least two high-sensitivity alternatives to achieve the best currently available in-gel protein detection: Sypro Ruby or Coomassie Blue.

Differential effects of targeted tongue exercise and treadmill running on aging tongue muscle structure and contractile properties

“…Each silver-stained gel was digitally imaged, and the optical density of each band was determined by investigators masked to rat age and treatment group using computer-assisted image analysis and densitometry (UN-Scan-IT gel version 6.1; Silk Scientific, Orem, UT). The ratio of the density of an individual myosin heavy chain (MHC) band to the total density within a column was used to determine the percentage of each MHC isoform per lane (14,25,27,32,68,76,84). Previous studies have demonstrated good to excellent reliability using these procedures (68).…”

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

“…Postelectrophoretic protein visualization is most frequently obtained by colorimetric and fluorescent staining methods. The critical factors of staining methods are their sensitivity, reproducibility and the linear range of detection (for a review see [97,98]). Colorimetric methods include CBB and variants, such as Colloidal (C)-CBB or blue silver (C-CBB modified), silver-staining (there are more than 100 variants) and zinc or imidazole-zinc staining.…”

“…Colorimetric methods include CBB and variants, such as Colloidal (C)-CBB or blue silver (C-CBB modified), silver-staining (there are more than 100 variants) and zinc or imidazole-zinc staining. Most of them have linear responses over a very limited range (maximum two orders of magnitude) due to saturation effects that make them unable to cover the great variation in protein concentration of a sample (see discussion reviewed by [97][98][99][100]). CBB-based stainings are simple to use and compatible with MS with a detection limit of around 8-100 ng/spot or 1-100 ng/spot for blue silver [101].…”

“…Fluorescence staining methods are being increasingly used due to their comparatively wide linear dynamic range (. 10 3 ), good sensitivity (1-2 ng protein/spot) and ease of use (for recent reviews [97,98]). The overall quality and relative cost of five commercially available fluorescent stains, Krypton, Deep Purple, Rubeo, Flamingo and the most popular used stain, Sypro Ruby dye were recently evaluated by Harris et al 2007 [97].…”

“…The overall quality and relative cost of five commercially available fluorescent stains, Krypton, Deep Purple, Rubeo, Flamingo and the most popular used stain, Sypro Ruby dye were recently evaluated by Harris et al 2007 [97]. The authors also reported that C-CBB was found to be comparable to Syrpo Ruby when detected using an infrared fluorescence imaging system rather than standard densitometry [97].…”