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

Journal of biosciences

Arnab Roy²  Umesh Varshney¹  Debnath Pal1² 

[1] Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India $$;Bioinformatics Centre, Supercomputer Education and Research Centre, Indian Institute of Science, Bangalore 560 012, India$$

关键词: Paper wick;    sample preparation;    sonication;    streaking;    two-dimensional electrophoresis;   

DOI  :  

来源: Indian Academy of Sciences

【 摘 要 】

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.

【 授权许可】

Unknown