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    • Avoiding acidic region streaking in two-dimensional gel electrophoresis: Case study with two bacterial whole cell protein extracts

      Arnab Roy Umesh Varshney Debnath Pal

      Articles Volume 39 Issue 4 September 2014 pp 631-642

    • Fulltext

       

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      Permanent link:
      https://www.ias.ac.in/article/fulltext/jbsc/039/04/0631-0642

    • Keywords

       

      Paper wick; sample preparation; sonication; streaking; two-dimensional electrophoresis

    • Abstract

       

      Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.

    • Author Affiliations

       

      Arnab Roy1 Umesh Varshney2 Debnath Pal1

      1. Bioinformatics Centre, Supercomputer Education and Research Centre, Indian Institute of Science, Bangalore 560 012, India
      2. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India

    • Dates

       
      Published
      September 2014
      Manuscript received
      14 February 2014
      Accepted
      June 2023

    • Supplementary Material

       

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