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三氯乙酸沉淀后进行样品超声处理可提高从培养的海马神经元中回收蛋白质的效率,并改善二维凝胶电泳的分辨率和重现性。

Sample sonication after trichloroacetic acid precipitation increases protein recovery from cultured hippocampal neurons, and improves resolution and reproducibility in two-dimensional gel electrophoresis.

作者信息

Manadas Bruno J, Vougas Konstantinos, Fountoulakis Michael, Duarte Carlos B

机构信息

Department of Zoology, Center for Neuroscience and Cell Biology, Dept. of Zoology, University of Coimbra, Coimbra, Portugal.

出版信息

Electrophoresis. 2006 May;27(9):1825-31. doi: 10.1002/elps.200500757.

Abstract

Protein precipitation with TCA followed by acetone washing is frequently used to clean samples before 2-DE. However, the difficulty in solubilizing TCA-precipitated proteins causes some variability in 2-D gels and makes it difficult to detect some proteins. In this work we show that sonication of the samples, after TCA precipitation followed by elution in sample buffer, increases total protein recovery, and improves reproducibility and matching ratios between gels when analyzed by specialized software.

摘要

在进行二维电泳(2-DE)之前,常用三氯乙酸(TCA)沉淀蛋白质,随后用丙酮洗涤来清理样品。然而,TCA沉淀的蛋白质难以溶解,这会导致二维凝胶出现一些差异,并且难以检测到某些蛋白质。在本研究中,我们表明,在TCA沉淀样品后,于样品缓冲液中洗脱,再对样品进行超声处理,可提高总蛋白回收率,并在使用专业软件分析时提高凝胶之间的重现性和匹配率。

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