Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels.
Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels.
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DOI：
10.3791/1431
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发表时间：
2009-08-03
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影响因子：
--
通讯作者：
Metzger, Sabine
中科院分区：
文献类型：
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作者：
Dyballa, Nadine;Metzger, Sabine
考马斯亮蓝（CBB）是一种常用于SDS-PAGE分离的蛋白质可视化的染料，提供了一个简单的染色程序和高定量。此外，它与质谱蛋白质鉴定完全兼容。但是，尽管有这些优点，CBB被认为比银或荧光染色灵敏度低，因此很少用于基于分析凝胶的蛋白质组学方法中的蛋白质检测。对原始考马斯方案（1）进行了几项改进，以提高CBB的灵敏度。引入了两个主要修改，以通过将染料分子转化为胶体颗粒来增强低丰度蛋白质的检测：1988年，Neuhoff及其同事将20%甲醇和更高浓度的硫酸铵应用于基于CBB G-250的染色溶液中（2），在2004年，Brachano等人在硫酸铵和甲醇的存在下使用CBB G-250与磷酸建立了蓝银（3）。然而，所有这些修饰仅允许检测约10 ng蛋白质。Kang等人在2002年发表了一个广泛不知名的胶体考马斯染色方案，其中他们修改了Neuhoff关于络合物质的胶体CBB染色方案。他们用硫酸铝代替硫酸铵，用毒性较小的乙醇代替甲醇（4）。Kang研究中的新型铝基染色显示出上级灵敏度，可检测低至1 ng/条带（磷酸化酶B），且灵敏度变化很小（取决于蛋白质）。在这里，我们展示了应用康的协议快速和敏感的胶体考马斯染色的蛋白质在分析的目的。我们将说明快速和简单的协议，使用二维凝胶常规进行我们的工作组。
Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches. Several improvements of the original Coomassie protocol(1) have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution(2), and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol(3). Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol(4). The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins. Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.