Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis
Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis
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DOI：
10.1002/elps.200305844
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发表时间：
2004-05-01
期刊：
影响因子：
2.9
通讯作者：
Righetti, PG
中科院分区：
文献类型：
--
作者：
Candiano, G;Bruschi, M;Righetti, PG
本文报道一种改良的纽霍夫胶体考马斯亮蓝G-250染色法，由于其灵敏度相当高，接近传统的银染色法，故被称为“蓝银”。与Neuhoff的方案相比，主要的修改是：染料浓度增加20%（从0.1%增加到0.12%），配方中磷酸的水平高得多（从2%增加到10%）。“蓝色银”显示出快得多的染料吸收（在着色的第一个小时期间为80%，而Sigma的商业制剂则没有）。即使在平衡（24小时染色）下，“蓝银”也表现出比所有其他配方高得多的灵敏度，接近（但低于）经典银染色的灵敏度。牛血清白蛋白（BSA）十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）后染色灵敏度的测量给出了在单个区带中1 ng的检测限（信噪比> 3）。与醛存在下的经典镀银方案相比，“蓝银”的灵敏度稍低，这在二维图谱分析后通过其与洗脱多肽链的质谱法的完全相容性得到充分补偿，从而证实没有染料共价结合（或永久修饰）到蛋白质材料中的任何残基。据信，配方中较高水平的磷酸，因此其较低的最终pH，有助于质子化多肽螺旋中最后解离的Asp和Glu残基，从而极大地有利于染料分子与蛋白质部分的离子锚定。然而，这样的结合必须伴随着与多肽主链上的芳香族和疏水残基沿着的相当大的疏水缔合。
A modified Neuhoff's colloidal Coomassie Blue G-250 stain is reported, dubbed "blue silver" on account of its considerably higher sensitivity, approaching the one of con- ventional silver staining. The main modifications, as compared to Neuhoff's protocol, were: a 20% increment in dye concentration (from 0.1 % up to 0.12%) and a much higher level of phosphoric acid in the recipe (from 2% up to 10%). The "blue silver" exhibits a much faster dye uptake (80% during the first hour of coloration, vs. none with a commercial preparation from Sigma). Even at equilibrium (24 h staining), the "blue silver" exhibits a much higher sensitivity than all other recipes, approaching (but lower than) the one of the classical silver stain. Measurements of stain sensitivity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of bovine serum albumin (BSA) gave a detection limit (signal-to-noise ratio > 3) of 1 ng in a single zone. The somewhat lower sensitivity of "blue silver" as compared to classical silvering protocols in the presence of aldehydes is amply compensated for by its full compatibility with mass spectrometry of eluted polypeptide chains, after a two-dimensional map analysis, thus confirming that no dye is covalently bound (or permanently modifies) to any residue in the proteinaceous material. It is believed that the higher level of phosphoric acid in the recipe, thus its lower final pH, helps in protonating the last dissociated residues of Asp and Glu in the polypeptide coils, thus greatly favoring ionic anchoring of dye molecules to the protein moiety. Such a binding, though, must be followed by considerable hydrophobic association with the aromatic and hydrophobic residues along the polypeptide backbone.