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Western Blotting pp 49–60Cite as

Solubilization of Proteins: The Importance of Lysis Buffer Choice

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1312))

Abstract

The efficient extraction of proteins of interest from cells and tissues is not always straightforward. Here we demonstrate the differences in extraction of the focal adhesion protein Kindlin-2 from choriocarcinoma cells using NP-40 and RIPA lysis buffer. Furthermore, we demonstrate the use of a more denaturing urea/thiourea lysis buffer for solubilization, by comparing its effectiveness for solubilization of small heat-shock proteins from smooth muscle with the often utilized RIPA lysis buffer. Overall, the results demonstrate the importance of establishing the optimal lysis buffer for specific protein solubilization within the experimental workflow.

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Acknowledgement

This work was supported by a Natural Sciences and Engineering Research Council Discovery Grant (#250218), an Establishment Grant from the Saskatchewan Health Research Foundation (SHRF; #2695), and a regional partnership program grant from SHRF (#2776) and the Canadian Institutes of Health Research (#ROP-101051) to D.J.M. M.P. and N.M. were holders of Alexander Graham Bell NSERC postgraduate fellowships.

Author information

Authors and Affiliations

  1. Division of BioMedical Sciences, Health Sciences Centre, Rm 5335, 300 Prince Philip Drive, St. John’s, NL, Canada, A1B 3V6

    Mandy Peach & Noelle Marsh

  2. Department of Veterinary Biomedical Sciences, One Reproductive Health Research Group, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Dr., Saskatoon, SK, Canada, S7N 5B4

    Ewa I. Miskiewicz & Daniel J. MacPhee

Authors
  1. Mandy Peach
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  2. Noelle Marsh
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  3. Ewa I. Miskiewicz
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  4. Daniel J. MacPhee
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Corresponding author

Correspondence to Daniel J. MacPhee .

Editor information

Editors and Affiliations

  1. Department of Medicine, University of Oklahoma, Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, Health Sciences Center, Department of Veterans, Oklahoma City, Oklahoma, USA

    Biji T. Kurien

  2. Department of Medicine, Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, Health Sciences Center, Department of Veterans Affairs Medical Center, Oklahoma City, Oklahoma, USA

    R. Hal Scofield

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Peach, M., Marsh, N., Miskiewicz, E.I., MacPhee, D.J. (2015). Solubilization of Proteins: The Importance of Lysis Buffer Choice. In: Kurien, B., Scofield, R. (eds) Western Blotting. Methods in Molecular Biology, vol 1312. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2694-7_8

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  • DOI: https://doi.org/10.1007/978-1-4939-2694-7_8

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2693-0

  • Online ISBN: 978-1-4939-2694-7

  • eBook Packages: Springer Protocols

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