Easy and Fast Western Blotting by Thin-Film Direct Coating with Suction

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• 作者：Chao-Yuan Liu ; De-Chao Lu ; Yi-Wei Jiang ; Yi-Kuang Yen ; Shih-Chung Chang ; An-Bang Wang
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：June 21, 2016
• 年：2016
• 卷：88
• 期：12
• 页码：6349-6356
• 全文大小：389K
• 年卷期：0
• ISSN：1520-6882

文摘

Thin-film direct coating (TDC) has been successfully used in Western blotting (WB). In this study, the advanced technique of TDC with suction (TDCS) was developed to reduce the consumption amount of antibody by a factor of up to 10⁴ in comparison with the amount consumed by the conventional WB using the capillary tube without any need of special micromachining processes. The operation time for completely finishing a high-quality WB can be reduced from 3 h in conventional WB to about 5 min or even less by TDCS. In addition, the signal-to-noise ratio of the immunoblotting by TDCS can be markedly increased. TDCS WB showed a high linearity within a 6-log₂ dynamic range for detecting 90–6000 ng of purified recombinant glutathione-S-transferase (GST) proteins and could particularly detect extrinsic GST proteins added in crude Escherichia coli or 293T cell lysates. Moreover, a protein mixture containing bovine serum albumin, GST, and ubiquitin could be specifically probed in parallel with their corresponding antibodies through multichannel TDCS WB. This simple and innovative TDCS WB offers various potential applications in simultaneously finishing multiple antibody–antigen screenings in a fast and single experiment.