The gel discs containing the protein of interest were washed twice with 50% ACN ＋ 50% ammonium bicarbonate (5 mM solution)，for a minimum of 2–3 h untill full decoloration of the gel. After that, wash twice (10 min each time) in 100% ACN. In-gel digestion was carried out in 100 mM ammonium bicarbonate, 1 mM CaCl2 pH 8.9, 30% ACN, and 12.5 ng/mL (1 mg) sequencing grade modified trypsin (Promega, Madison, WI, USA) overnight at 37 C.
至于用Ｇ-250的原因我觉得主要是：
Fazekas de St. Groth , have always enjoyed a widespread popularity, due to their ease of use and reasonable sensitivity (ca. 0.5 mg/mm2). One mg protein can bind
0.17 mg of Amido black, 0.23 mg of Fast Green, 1.2 mg of Coomassie Blue R-250 (R = reddish hue) and 1.4 mg of Coomassie Blue G-250 (G = greenish hue)
回答：
１ 硫酸氨的溶解度很大，在20溶解度为75.3克/100毫升,80度时为95.3克/100毫升,100度时为103克/100毫升,故在常温下单纯100毫升水很难能溶解100克的,溶液早已呈饱和状态;而加入100毫升磷酸会不会帮助硫酸氨的溶解就不得而知了,既然原文中两次提到"hen the ammonium sulfate has dissolved, add enough Coomassie Blue G-250"和"When all olids have dissolved,add water to 80% of the final volume"那必有他的原因,文中是将硫酸氨加入水和磷酸混合液中,楼主可以试试这样缓慢加入会不会有助于溶解,大家关注这种方法的话也可以试试嘛
2 乙醇应该可以替代甲醇的,但究竟这个替代对灵敏度会不会有影响就不清楚了;
3 磷酸偶觉得还是85%就当85%用,"the desired amount of phosphoric acid is added, so that, in the final volume, its concentration ill be 10%"在终体积下浓度为10%,既然本法的关键所在便是提高磷酸的浓度,那"分析纯85％的磷酸当100％的来配液体"势必达不到最佳的浓度,所以我觉得楼主的染色灵敏度还可以再提高些些;
我找到一个protocol（cuturl('http://www.proteomics.missouri.edu/')）又有点不同：
Colloidal Coomassie Brilliant Blue (G-250) – slightly better sensitivity than R-250 and is compatible with mass spectrometric analysis.
1. Wash SDS from gel with three consecutive washes (10 min each) in MilliQ water. Longer washes may be necessary for 18 and 24 cm gels. It is critical to remove residual SDS from gel prior to staining.
2. Add enough Colloidal Coomassie stain (20% ethanol, 1.6% phosphoric acid, 8% ammonium sulfate, 0.08% Coomassie Brilliant Blue G-250) to cover gel.
3. Incubate at room temperature on rotary agitator for at least six hours.
4. Decant stain and rinse gel twice with MilliQ water (1 min each).
5. Destain gel with MilliQ water until background is low (4 hours).
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