Evaluation of band sharpness, resolution and sensitivity
The band resolution and sensitivity of the three staining procedures were evaluated. For the evaluation of the resolution of the staining procedure, a total cell protein extract from late dough stage Taichung 65 rice endosperm was used. Rice endosperms were ground into powder and proteins were extracted using imidazole-HCl buffer . The extracted proteins were quantified using the Bradford assay . Then, 20.0 µg and 6.7 µg of protein samples were run on SDS-PAGE. For evaluation of the staining sensitivity, bovine albumin (BA) (Amresco) was used. The BA stock solution of 10 mg/mL was prepared. The stock solution was diluted to a series of concentrations and the following amounts of BA were run on SDS-PAGE: 2000.0 ng, 666.7 ng, 222.2 ng, 74.1 ng, 24.7 ng, and 8.2 ng.
SDS-PAGE was run using the standard method by Laemmli , using 10% separating gels and 4% stacking gels, and a 1 mm-thick mini-gel format with 0.5-cm wells [Biorad Mini Protean II system]. Gels were run at 90 V for 30 min, then 150 V for about 60 min or until the dye front reached the end of the gel. After SDS-PAGE, the gels were stained with one of the three staining methods described henceforth. Triplicate gels were run for each staining method.
The improved colloidal CBB-G method was also evaluated for MS compatibility. Rice endosperm protein extracts were subjected to isoelectric focusing using non-linear pH 3–10 IPG strips (Biorad #1632016). After focusing, IPG strips were equilibrated, and the second dimension was run using 11% mini-gels. After gel imaging and analysis, 49 differentially expressed spots were selected for MS identification. Each spot was pooled from three to six gels, excised, trypsin digested and analysed using LC–MS/MS (Thermo™ Q Exactive Plus Hybrid Quadrupole-Orbitrap).
Standard colloidal CBB-G staining method
The standard colloidal CBB-G staining method was performed as described by Dyballa and Metzger . After SDS-PAGE, the gel was rinsed three times with ultrapure water by shaking on a platform shaker at 80 rpm for 10 min each time. Then, the ultrapure water was decanted and the gel was incubated in CBB-G staining solution 0.02% (w/v) CBB G-250 (Sigma), 5% (w/v) aluminium sulfate (Bendosen), 10% (v/v) ethanol (HmbG), 2% (v/v) orthophosphoric acid (Merck) for 2 h with shaking at 80 rpm, or overnight, with or without shaking. If the staining solution turned a bright blue, the staining solution was replaced with a fresh solution. For maximum sensitivity, gels were stained overnight.
For destaining, the gels were rinsed twice with ultrapure water, and destained in CBB-G destain solution (10% ethanol, 2% orthophosphoric acid) for 10–60 min with shaking. After that, the gels were rinsed twice with ultrapure water.
The improved colloidal CBB-G staining method
For the improved colloidal CBB-G method, the staining protocol by Dyballa and Metzger  was slightly modified with an additional fixation step and a simplified destaining step. After the SDS-PAGE run, the gel was transferred to a plastic box and fixed with fixation solution [40% methanol (Merck), 10% acetic acid (Bendosen)] for 30 min, with shaking at 80 rpm. For convenience, this fixation step could be extended overnight or up to several days. After fixation, the gel was rinsed briefly with ultrapure water. The subsequent steps followed the colloidal CBB-G stain protocol described previously. Figure 2 illustrates the differences between the improved and standard protocols.
For the destaining process, the gel was rinsed briefly with ultrapure water, then destained in CBB-G destain solution by shaking on a platform shaker at 80 rpm for roughly 3–5 min. The gel was then rinsed briefly with ultrapure water, then washed with ultrapure water by shaking on a platform shaker at 80 rpm for 10 min. The water was then decanted, and the gel was briefly rinsed till all colloidal particles were removed from the staining box as colloidal particles may interfere with gel imaging. The gels were stored in ultrapure water at 4 °C.
For the CBB-R staining method, a standard staining protocol was used as shown in Fig. 3 (Bio-Rad manual). Briefly, the gel was immersed in CBB-R staining solution [0.1% CBB R-250 (Bio-Rad), 40% methanol, 10% acetic acid] overnight. The following morning, the gel was rinsed with water and destained with CBB-R destaining solution (40% methanol, 10% acetic acid) by shaking on a platform shaker at 80 rpm for several hours, with two to three changes of destaining solution.
Evaluation of resolution and sensitivity
Evaluation of resolution was conducted by visual observation of gels as well as through analysis of plot profiles generated using ImageJ software . For sensitivity analysis, the presence of the BA bands were indicated by a distinct peak at the correct position in the plot profile. The areas under the peak (corresponding to band intensity) were calculated, and statistical analysis was conducted using the unpaired t-test.
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