Comparing the effects of different protocols on the destaining of the gel background and detection of BSA bands.

<p>The effects of different protocols on gel background destaining was compared: gels were stained according to Dong <i>et al</i>. and destained six times for 1 min in boiling water, according to the original protocol (A); gels were stained according to Dong <i>et al</i>. and destained for 1 hr in boiling water (B); gels were stained according to Dong <i>et al</i>. 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 CBS, and destained for 1 hr in a boiling EDTA solution (D). Gel background intensities were compared for all tested protocols (E), the results are expressed as mean values ± standard deviations from three independent experiments ; a, b, c, and d correspond to the above mentioned protocols A, B, C, and D, respectively. To estimate the differences in detection of BSA bands in gels stained according to the protocols A (blue), B (red), C (green), and D (purple), corresponding band areas were measured (F); the inset shows more detail illustration for lanes 5–8. The results are expressed as mean values ± standard deviations from three independent experiments. Two-fold serially diluted BSA samples were used for 1D SDS-PAGE and corresponded to 1000, 500, 250, 125, 62.5, 31.25, 15.6, and 7.8 ng per lanes 1–8, respectively.</p

Staining of different protein fractions.

<p>1D SDS-PAGE gels were stained according to the original Dong protocol (lanes 1) and according to our new proposed protocol with imidazole-zinc reverse staining followed by fast CBS and EDTA destaining (lanes 2) to illustrate the effect of both protocols. To compare various protein types four different peripheral blood mononuclear cell protein fractions were used: cytosolic (A), nuclear (B), membrane (C), and cytoskeleton (D) protein fractions. Brightness and contrast of the magnified insets were adjusted for better illustrations.</p

A comparison of bacterial (<i>E. coli</i>) proteins staining.

<p>Whole cell lysate (<i>E. coli</i>) was separated by 1D SDS-PAGE and gels were stained according to the original Dong protocol (lane 1) and according to our new proposed protocol with imidazole-zinc reverse staining followed by fast CBS and EDTA destaining (lane 2) to illustrate the effect of both protocols. Brightness and contrast of the magnified insets were adjusted for better illustrations.</p

The influence of EDTA concentration on gel destaining.

<p>Illustrations of gel backgrounds after 1 hr incubation using different EDTA concentrations (0, 0.5, 1, 2, 4, 8, 16, 32, and 40 mM EDTA solutions, respectively) are shown (A). Average area intensity values and their standard deviations for gels destained with corresponding EDTA concentrations are presented as estimated using ImageJ software (B).</p

2D SDS-PAGE of blood platelet, undepleted plasma, and rat brain tissue samples.

<p>The effect of an appropriate protocol on gel background destaining and spot detection is illustrated using blood platelet, undepleted plasma, and rat brain tissue samples analyzed by 2D SDS-PAGE. The gels were processed according to the original Dong (Protocol A) and the final (Protocol D) protocols. Magnified insets were exported from the Progenesis SameSpots software (brightness and contrast of the insets were adjusted by the software) and correspond to the gray areas highlighted in the gels. Spots that were detected in gels stained according to the final protocol D only are indicated with circles or arrows.</p

The influence of temperature on gel destaining.

<p>Gels were destained with a boiling EDTA destaining solution for 1 hr (1); the combination of six washes in a boiling EDTA solution for 1 min followed by a 1 hr room temperature EDTA washing solution (2); a room temperature EDTA washing solution for 1 hr (3) or overnight (4); and the combination of six washes in a boiling EDTA solution for 1 min followed by a 6 hr room temperature washing (5). Illustrations of gel backgrounds after destaining with appropriate procedures are shown (A). Average area intensity values and their standard deviations for gels destained using appropriate procedures are presented as estimated using ImageJ software (B).</p