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Effective Solubilization Procedure for Analysis of Silkworm Hemolymph Proteins by Two-Dimensional Gel Electrophoresis

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Abstract

The outstanding capability of two-dimensional gel electrophoresis in separating all types of proteins basically depends on the efficiency of sample preparation. Sample preparation is one of the most critical steps in two-dimensional gel electrophoresis. Unfortunately, due to severe solubility, resolution of protein on gel is usually hampered, and thus, analysis remains a difficult task. However, technically several problems are generally encountered during protein extraction and isoelectric focusing. In the present investigation, we emphasized on evaluation and comparison of six different protein solubilization methods intended for resolving and analyzing silkworm hemolymph proteins by two-dimensional gel electrophoresis. Our findings revealed that the buffer composition of 8 M urea, 4 % 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 40 mM Tris base, 65 mM dithiothreitol, and 0.2 % ampholyte can effectively solubilize and yields maximum protein spots.

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Acknowledgments

Dr. Shyam Kumar would like to acknowledge the financial support granted by University Grants Commission New Delhi, India for the present research (grant no. F. 36-139/2008 (SR) dated 27th March 2009) and thanks to CSGRC, Hosur for providing silkworm races for this research work.

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Authors and Affiliations

  1. Department of Sericulture, Karnatak University, Dharwad, 580 003, Karnataka, India

    Mallikarjun Gadwala

  2. Department of Zoology, Karnatak University, Dharwad, 580 003, Karnataka, India

    Neetha Kari

  3. Institute of Agricultural Biotechnology, University of Agricultural Sciences, Dharwad, 580 008, Karnataka, India

    Narayan Moger

  4. Department of Biotechnology and Microbiology, Karnatak University, Dharwad, 580 003, Karnataka, India

    Shyam Kumar Vootla

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  1. Mallikarjun Gadwala
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  2. Neetha Kari
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  3. Narayan Moger
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  4. Shyam Kumar Vootla
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Correspondence to Shyam Kumar Vootla.

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Gadwala, M., Kari, N., Moger, N. et al. Effective Solubilization Procedure for Analysis of Silkworm Hemolymph Proteins by Two-Dimensional Gel Electrophoresis. Appl Biochem Biotechnol 169, 1459–1466 (2013). https://doi.org/10.1007/s12010-012-0067-0

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  • DOI: https://doi.org/10.1007/s12010-012-0067-0

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