Improved Coomassie Blue Dye-Based Fast Staining Protocol for Proteins Separated by SDS-PAGE

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.

The effect of the biological variability of samples on Coomassie blue dye based fast staining for SDS‐PAGE in nonfixed gels

“…fast‐staining protocol (protocol A) or according to the improved fast‐staining protocol by Májek et al. (protocol B). Briefly, fast‐staining protocol A workflow was as follows: immediately after electrophoresis, the gels were boiled in water for 1 min; stained for 2 min with 0.05% w/v Coomassie blue in water at boiling temperature; and destained with boiling water for 1 min each for six times.…”

“…All 2D electropherograms were processed using Progenesis SameSpots software (Nonlinear Dynamics, Newcastle upon Tyne, UK); samples (n = 5) were compared separately and differences in the proteome patterns of each sample (triplicate gels stained according to the appropriate protocol) were further mutually confronted. The most obvious change that was observed corresponded to the spot that had been identified as a missing spot in our previous work [4]. Surprisingly, significant discoloration (near to the phenomenon of a missing spot) of this spot was observed for only one of the five samples, while no obvious change was observed for the rest of samples; spot discoloration should not be confused with a reduction in spot intensity, as caused by the gel staining with the less sensitive standard fast-staining protocol A, as reported previously [3].…”

“…Therefore, the nonfixed fast-staining approach should be used only for Table 1-r sA /r sB (V1/S1) of sample 4) was used for the comparison of different fixing and staining conditions. Gels were stained according to the protocol A without (1) or with (2) the boiling water fixation step, according to the improved protocol B (3), and using 10% acetic acid/30% methanol fixation for 30 min before staining according to the protocol A followed by water (4) or EDTA (5) destaining.…”

“…This should be considered even if using pooled plasma samples, as variability of samples may still occur [8]. To avoid eventual detection issues, the improved fast-staining protocol B by Májek et al [4] based on reverse imidazole-zinc staining can be used or another protein fixation step should be included.…”

A new procedure for fast soft staining of BN‐PAGEs on photosynthetic complexes

“…Since the time for staining is strictly dependent on the gel thickness, polyacrylamide composition, and temperature , it was established that the most suitable procedure needs an overnight staining at room temperature . In general, under an acidic environment, the staining procedure using CBB improves the ionic interactions with the basic amino acid residues of the proteins and increases secondary interactions via van der Waals attraction, hydrogen bonding, and hydrophobic interactions, improving significantly the contrast . However, in BN‐PAGEs, the use of CBB was introduced not only because of its property of tightly binding to the proteins, but also because of its ability to induce a surface density of negative charge on the protein, enhancing the propensity of the proteins to stay in solution and to move in an electric field.…”