Comparison of fluorescent stains: Relative photostability and differential staining of proteins in two‐dimensional gels

Abstract

The fluorescence of proteins stained with Deep Purple and SYPRO Ruby was measured over a time course of UV transillumination to determine the relative photostability of each stain. Mean spot fluorescence (n = 200 matched spots) in gels stained with Deep Purple decreased 27% following 2 min of UV transillumination, compared to SYPRO Ruby, which decreased 17%. After 19 min, an 83% decrease in Deep Purple fluorescence was observed, compared to 44% for SYPRO Ruby. By interpolation, the half-life of Deep Purple fluorescence was estimated to be approximately 6 min. The half-life of SYPRO Ruby fluorescence was not reached during the 19 min time course. Further, differential staining of proteins was observed in gels stained with Deep Purple and SYPRO Ruby as compared to colloidal Coomassie Brilliant Blue and silver staining.

Statistical Model to Analyze Quantitative Proteomics Data Obtained by 18O/16O Labeling and Linear Ion Trap Mass Spectrometry

“…In these studies two or more protein extracts from tissues, cells, or body fluids in changing environmental or physiological conditions are assayed to determine the presence of proteins exhibiting an alteration in their expression levels. Proteins have traditionally been separated, visualized, and subjected to relative quantification by two-dimensional gel electrophoresis and protein poststaining (3,4) or prelabeling (5,6). The spots showing quantitative differences were then excised and proteolyzed, and the resulting peptides were used to identify the proteins by MS (7).…”

Experimental and Statistical Considerations to Avoid False Conclusions in Proteomics Studies Using Differential In-gel Electrophoresis

“…The resulting spot patterns can be visualized by labeling the sample prior to the separation, e.g. with radioactivity (21) or fluorescence (1,6,22), or after the separation with total protein stains such as colloidal Coomassie (2), SYPRO Ruby, or Deep Purple (23,24). The spot volumes can be compared from one sample to another, and it is an established technique in expression studies.…”

The Coomassie chronicles: past, present and future perspectives in polyacrylamide gel staining

“…The ruthenium ion co-ordinates three bathrophenanthroline disulfonates resulting in a hexasulfonated complex that avidly binds proteins in their protonated state. A correlative of 0.83 was found between SYPRO Ruby and CBB staining [25].…”

“…Moreover, silver staining correlates poorly with other stains. In a recent comparison of 944 proteins matched from 2D gels, the correlation coefficient of silver staining with SYPRO ® Ruby and CBB staining was 0.48 and 0.66, respectively [25].…”

“…As a consequence, residual epicoccone in the gel is also converted in the process, which generates background fluorescence and decreases the signal-to-noise ratio. Another disadvantage of the stain is its photoinstability [25]. The primary mechanism of epicocconone binding is through hydrophobic interaction with the lypophilic tail of SDS bound to the protein, similar to the mechanisms of Nile Red [48] and the SYPRO Red and Orange dyes [49].…”