Vettermann Christian, Jäck Hans-Martin, Mielenz Dirk
Division of Molecular Immunology, Department of Internal Medicine III, Nikolaus Fiebiger Center for Molecular Medicine, University of Erlangen-Nürnberg, 91054 Erlangen, Germany.
Anal Biochem. 2002 Sep 15;308(2):381-7. doi: 10.1016/s0003-2697(02)00269-5.
The characterization of protein expression patterns by two-dimensional gel electrophoresis depends on efficient and reliable identification strategies for target spots. In addition to sophisticated techniques, such as microsequencing and peptide mass spectrometry, immunodetection of membrane-immobilized proteins is a valuable method with which to identify the corresponding spots for a given set of candidate proteins. To precisely assign immunoreactive spots, this approach requires specific immunodetection and staining of total protein to be performed on the same membrane. Here, we describe a highly sensitive, colloidal silver-based method for the assignment of immunoreactive spots in two-dimensional protein patterns. This simple and rapid procedure involves a destaining step after staining of nitrocellulose-bound proteins with colloidal silver. We show that destaining of proteins is a prerequisite for subsequent immunodetection using enhanced chemiluminescence. Several types of antibodies were successfully employed for antigen detection after the staining-destaining procedure. Our results demonstrate that the colloidal silver-based method is generally applicable for the unambiguous identification of candidate proteins in complex two-dimensional patterns.
通过二维凝胶电泳对蛋白质表达模式进行表征，依赖于针对目标斑点的高效且可靠的鉴定策略。除了诸如微量测序和肽质谱分析等复杂技术外，对膜固定蛋白进行免疫检测是一种有价值的方法，可用于识别给定候选蛋白集的相应斑点。为了精确确定免疫反应性斑点，这种方法需要在同一膜上对总蛋白进行特异性免疫检测和染色。在此，我们描述了一种基于胶体银的高灵敏度方法，用于在二维蛋白质模式中确定免疫反应性斑点。这个简单快速的过程包括在用胶体银对硝酸纤维素结合蛋白进行染色后进行脱色步骤。我们表明，蛋白质脱色是随后使用增强化学发光进行免疫检测的先决条件。在染色 - 脱色程序后，几种类型的抗体成功用于抗原检测。我们的结果表明，基于胶体银的方法通常适用于在复杂的二维模式中明确鉴定候选蛋白。
