Central Drug Research Institute, Lucknow, India.
Electrophoresis. 2012 Oct;33(19-20):3062-5. doi: 10.1002/elps.201200333. Epub 2012 Sep 24.
Western blotting is a widely used analytical technique for detection of specific protein(s) in a given sample of tissue/cell homogenate or extract. Both chemiluminescence (CL) and colorimetric detections can be used for imaging Western blots. Colorimetric substrates offer background free, sensitive, and clean imaging results directly on the blotted membrane and provides more accurate profile with respect to prestained marker. However, blots stained with colorimetric substrates cannot be reused since no stripping protocols have been reported for such blots, thus limiting their reuse for detection of another protein. In the present study, for the first time, we report a novel method of stripping Western blots developed with the colorimetric substrate TMB for detection of a low-abundant protein and reprobing of these blots after stripping for detection of a more abundant protein through CL procedure. The stripping procedure utilizes a stripping buffer consisting of β-mercaptoethanol, SDS, and Tris-HCl and a washing buffer consisting of PBS added with 0.1% Tween-20 involves a series of steps and facilitates accurate detection of the second protein (i.e., more abundant protein) in the stripped blot through CL. The protocol is reproducible and facilitates saving of precious clinical samples, in addition to saving cost and time as compared to the existing procedures.
蛋白质印迹法是一种广泛用于检测组织/细胞匀浆或提取物中特定蛋白质的分析技术。化学发光（CL）和比色检测均可用于对 Western 印迹进行成像。比色底物提供无背景、敏感且清洁的成像结果，直接在印迹膜上进行，并且相对于预染标记物提供更准确的谱图。然而，由于没有报道用于这种印迹的剥离方案，因此用比色底物染色的印迹不能重复使用，从而限制了它们在检测另一种蛋白质时的重复使用。在本研究中，我们首次报道了一种新的方法，即使用比色底物 TMB 对 Western 印迹进行剥离，用于检测低丰度蛋白质，并在剥离后通过 CL 程序重新探测这些印迹以检测更丰富的蛋白质。该剥离程序使用含有β-巯基乙醇、SDS 和 Tris-HCl 的剥离缓冲液和含有 0.1%吐温-20 的 PBS 洗涤缓冲液，涉及一系列步骤，可通过 CL 准确检测剥离印迹中的第二种蛋白质（即更丰富的蛋白质）。该方案具有可重复性，除了与现有程序相比节省成本和时间外，还可以节省宝贵的临床样本。
