Assessing detection methods for gel-based proteomic analyses.
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.
Harris
Lauren R
LR
Department of Physiology & Biophysics, Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, AB, T2N 4N1, Canada.
Churchward
Matthew A
MA
Butt
R Hussain
RH
Coorssen
Jens R
JR
D000818:Animals; D004396:Coloring Agents / Q000737:chemistry / Q000592:standards*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000592:standards*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000592:standards*; D051379:Mice; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000592:standards*; D012680:Sensitivity and Specificity
D004396:Coloring Agents; D005456:Fluorescent Dyes; D011506:Proteins