Abstract
The time required to visualize proteins using Coomassie Blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a Coomassie Blue dye solution at boiling temperatures. However, fast stainings suffer from high gel backgrounds, reducing the signal-to-noise ratio and limiting the number of detectable spots in the case of 2D SDS-PAGE. The aim of this work was to eliminate the high gel background, and thus improve fast staining protocols based on Coomassie Blue dye. We show that merely replacing water with a 4 mM EDTA washing solution at boiling temperatures, results in a transparent gel background within 50 to 60 minutes of destaining. Moreover, when a combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining is used the sensitivity is improved significantly; nanogram amounts of proteins can be detected using 1D SDS-PAGE, and about 30% to 60% more spots can be detected with 2D SDS-PAGE in plasma, platelet, and rat brain tissue samples. This work represents an optimized fast staining protocol with improved sensitivity, requiring between 60 to 75 minutes to complete protein visualization.
Highlights
The visualization of proteins separated by SDS-PAGE is one of the crucial steps in gel-based proteomics
The effects of different protocols on gel background destaining was compared: gels were stained according to Dong et al and destained six times for 1 min in boiling water, according to the original protocol (A); gels were stained according to Dong et al and destained for 1 hr in boiling water (B); gels were stained according to Dong et al and destained for 1 hr in a boiling EDTA solution (C); and gels were stained according to our new proposed protocol with imidazole-zinc reverse staining followed by fast Coomassie Blue staining (CBS), and destained for 1 hr in a boiling EDTA solution (D)
It has been shown that a high gel background is the limiting factor of fast CBS [4]
Summary
The visualization of proteins separated by SDS-PAGE is one of the crucial steps in gel-based proteomics. There are other staining protocols more sensitive than CBS [1,2]; and using Coomassie Blue dye can be time consuming. The time required to visualize proteins using CBS has been significantly reduced with fast staining protocols utilizing Coomassie Blue dye. A protocol introduced by Dong et al enabled simple protein visualization, requiring only minutes to finish the staining procedure [3]. We aimed to establish a fast staining protocol that would address the gel background issue and retain the advantages of a fast procedure based on Dong’s protocol. We present an optimized fast staining protocol with improved sensitivity, which requires just 60 to 75 minutes to complete protein visualization
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