Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Biosciences and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou Hainan, PR China.
Electrophoresis. 2012 Jan;33(2):296-306. doi: 10.1002/elps.201100300.
Considering the importance of CBB staining in visualizing proteins in 2-DE gels, any improvement in the existing protocols with high sensitivity and good MS compatibility is of significant importance. In this study, we systematically evaluated the effects of different staining parameters on CBB methods by 1-DE and 2-DE, and demonstrated that G-250 was more suitable for visualizing low-abundant proteins as well as generating more spots than R-250, whereas R-250 had a superior capability for quick staining of high-abundant proteins. The staining produced by mixing G-250 and R-250 in different ratios showed similar sensitivity. Compared with acetic acid, phosphoric acid produced more protein spots. Ammonium-based stain demonstrated a superior sensitivity than the aluminum-based one. Based on these findings, a new protocol using CBB G-250, ammonium sulfate and phosphoric acid (GAP) was developed by incorporating the fixation, sensitization and staining procedures together. The comparison of GAP with other methods revealed that GAP generated more protein spots and had wider applications. The identification of 11 proteins demonstrated that GAP was not only compatible with MS but also obviously reduced in vitro protein modification, and thus could be a preferable protocol in the future proteomic analysis.
鉴于 CBB 染色在可视化 2-DE 凝胶中的蛋白质的重要性，任何提高现有方法的灵敏度和良好的 MS 兼容性的改进都具有重要意义。在这项研究中，我们通过 1-DE 和 2-DE 系统地评估了不同染色参数对 CBB 方法的影响，结果表明 G-250 比 R-250 更适合可视化低丰度蛋白质并产生更多斑点，而 R-250 则更适合快速染色高丰度蛋白质。G-250 和 R-250 以不同比例混合染色的效果相似。与乙酸相比，磷酸产生的蛋白质斑点更多。基于氨的染色比基于铝的染色具有更高的灵敏度。基于这些发现，通过将固定、敏化和染色过程结合在一起，开发了一种使用 CBB G-250、硫酸铵和磷酸（GAP）的新方案。将 GAP 与其他方法进行比较表明，GAP 产生的蛋白质斑点更多，应用范围更广。对 11 种蛋白质的鉴定表明，GAP 不仅与 MS 兼容，而且明显减少了体外蛋白质修饰，因此在未来的蛋白质组学分析中可能是一种更可取的方案。
