Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues

Norio Yamamoto1, Yoko Yamashita2, Yasukiyo Yoshioka3, Shin Nishiumi4, Hitoshi Ashida2

1 Research & Development Institute, House Wellness Foods Corporation, Itami, 2 Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, Kobe, 3 Organization of Advanced Science and Technology, Kobe University, Kobe, 4 Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.18
DOI:  10.1002/cpps.13
Online Posting Date:  August, 2016
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Abstract

Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane‐embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc.

Keywords: membrane protein; membrane fraction; plasma membrane; protein extraction; western blotting

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of a Plasma Membrane Fraction from Cultured Muscle Cells or Muscle Tissue
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of a Plasma Membrane Fraction from Cultured Muscle Cells or Muscle Tissue

  Materials
  • Cultured muscle cells: e.g., L6 rat muscle cells (ATCC, cat. no. CRL‐1458) or C2C12 mouse muscle cells (ATCC, cat no. CRL‐1772)
  • Cultured adipose cells: e.g., 3T3‐L1 mouse adipose cells (ATCC, cat. no. CL‐173)
  • Fresh or deep‐frozen muscle or adipose tissue: e.g., soreus muscle of hind limb or epididymal adipose tissue
  • Appropriate culture medium (e.g., MEM for L6 cells or DMEM for C2C12 cells and 3T3‐L1 cells) supplemented with FBS and antibiotics
  • PBS, ice cold ( appendix 2E)
  • Buffer A containing 0.1% (v/v) NP‐40 (see recipe)
  • Buffer A containing 1% (v/v) NP‐40 (see recipe)
  • 2× RIPA lysis buffer (see recipe)
  • 60‐mm culture dish, collagen‐coated (Corning, cat. no. 354401)
  • Cell scrapers (Thermo Scientific, cat. no. 179693)
  • Dissecting scissors, straight, sharp‐tip
  • 1.5‐ml microcentrifuge tube and pestle combo (Argos Technologies, cat. no. P9950‐901)
  • Handheld pestle motor mixer (Fisher Science, cat. no. 12‐141‐361)
  • 22‐gauge syringe needle (Terumo, cat. no. NN‐2225R)
  • 1‐ml disposable syringe (Terumo, cat. no. SS‐01 T)
  • Microcentrifuge, preferably with refrigeration
  • Additional reagents and equipment for protein assay (unit 3.4; Olson and Markwell, ), SDS gel electrophoresis, electroblotting of protein gels, and detection of proteins on blot membranes (units 10.1, 10.7, and 10.8; Gallagher, 29.18; Goldman et al., 29.18; Harper and Speicher, 29.18)
NOTE: All solutions and equipment that come into contact with cells must be sterile, and appropriate aseptic technique must be used when working with cell cultures.NOTE: Keep samples at 0° to 4°C, and perform all centrifugation steps at 4°C in a cold room or in a refrigerated microcentrifuge.
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Figures

Videos

Literature Cited

  Abel, E.D., Shepherd, P.R., and Kahn, B.B. 2004. Glucose transporters and pathophysiologic states. In Diabetes Mellitus: A Fundamental and Clinical Text, 3rd ed. (D. LeRoith, S.I. Taylor, and J.M. Olefsky, eds.) pp. 917‐938. Lipincott Williams & Wilkins, Philadelphia.
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  Asahi, Y., Hayashi, H., Wang, L., and Ebina, Y. 1999. Fluoromicroscopic detection of myc‐tagged GLUT4 on the cell surface. Co‐localization of the translocated GLUT4 with rearranged actin by insulin treatment in CHO cells and L6 myotubes. J. Med. Invest. 46:192‐199. PMID: 10687315.
  Bünger, S., Roblick, U.J., and Habermann, J.K. 2009. Comparison of five commercial extraction kits for subsequent membrane protein profiling. Cytotechnology 61:153‐159. doi: 10.1007/s10616‐009‐9249‐1.
  Cooper, G.M. 2000. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; Structure of the Plasma Membrane. Available from: http://www.ncbi.nlm.nih.gov/books/NBK9898/.
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  Dobson, S.P., Livingstone, C., Gould, G.W., and Tavaré, J.M. 1996. Dynamics of insulin‐stimulated translocation of GLUT4 in single living cells visualised using green fluorescent protein. FEBS Lett. 393:179‐184. PMID: 8814285 doi: 10.1016/0014‐5793(96)00879‐4.
  Fukumoto, H., Kayano, T., Buse, J.B., Edwards, Y., Pilch, P.F., Bell, G.I., and Seino, S. 1989. Cloning and characterization of the major insulin‐responsive glucose transporter expressed in human skeletal muscle and other insulin‐responsive tissues. J. Biol. Chem. 264:7776‐7779. PMID: 2656669.
  Gallagher, S.R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Protein Sci. 68:10.1.1‐10.1.44. doi: 10.1002/0471140864.ps1001s68.
  Goldman, A., Ursitti, J.A., Mozdzanowski, J., and Speicher, D.W. 2015. Electroblotting from polyacrylamide gels. Curr. Protoc. Protein Sci. 82:10.7.1‐10.7.16. doi: 10.1002/0471140864.ps1007s82.
  Harper, S. and Speicher, D.W. 2001. Detection of proteins on blot membranes. Curr. Protoc. Protein Sci. 00:10.8.1‐10.8.7.
  Itagaki, K., Naito, T., Iwakiri, R., Haga, M., Miura, S., Saito, Y., Owaki, T., Kamiya, S., Iyoda, T., Yajima, H., Iwashita, S., Ejiri, S., and Fukai, F. 2012. Eukaryotic translation elongation factor 1A induces anoikis by triggering cell detachment. J. Biol. Chem. 287:16037‐16046. doi: 10.1074/jbc.M111.308122.
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  Lin, S.H. and Guidotti, G. 2009. Purification of membrane proteins. Methods Enzymol. 463:619‐629. doi: 10.1016/S0076‐6879(09)63035‐4.
  Nagano, T., Hayashibara, K., Ueda‐Wakagi, M., Yamashita, Y., and Ashida, H. 2015. Black tea polyphenols promotes GLUT4 translocation through both PI3K‐and AMPK‐dependent pathways in skeletal muscle cells. Food Sci. Technol. Res. 21:489‐494. doi: 10.3136/fstr.21.489.
  Nishiumi, S. and Ashida, H. 2007. Rapid preparation of a plasma membrane fraction from adipocytes and muscle cells: Application to detection of translocated glucose transporter 4 on the plasma membrane. Biosci. Biotechnol. Biochem. 71:2343‐2346. doi: 10.1271/bbb.70342.
  Oh, Y., Jeon, Y.J., Hong, G.S., Kim, I., Woo, H.N., and Jung, Y.K. 2012. Regulation in the targeting of TRAIL receptor 1 to cell surface via GODZ for TRAIL sensitivity in tumor cells. Cell Death Differ. 19:1196‐1207. doi: 10.1038/cdd.2011.209.
  Olson, B.J. and Markwell, J. 2007. Assays for determination of protein concentration. Curr. Protoc. Protein Sci. 48:3.4.1‐3.4.29.
  Yamamoto, N. and Ashida, H. 2012. Evaluation methods for facilitative glucose transport in cells and their applications. Food Sci. Technol. Res. 18:493‐503. doi: 10.3136/fstr.18.493.
  Yamamoto, N., Kawabata, K., Sawada, K., Ueda, M., Fukuda, I., Kawasaki, K., Murakami, A., and Ashida, H. 2011. Cardamonin stimulates glucose uptake through translocation of glucose transporter‐4 in L6 myotubes. Phytother. Res. 25:1218‐1224. doi: 10.1002/ptr.3416.
  Yamamoto, N., Ueda‐Wakagi, M., Sato, T., Kawasaki, K., Sawada, K., Kawabata, K., Akagawa, M., and Ashida, H. 2015. Measurement of glucose uptake in cultured cells. Curr. Protoc. Pharmacol. 71:12.14.1‐12.14.26. doi: 10.1002/0471141755.ph1214s71.
  Yamashita, Y., Okabe, M., Natsume, M., and Ashida, H. 2012. Prevention mechanisms of glucose intolerance and obesity by cacao liquor procyanidin extract in high‐fat diet‐fed C57BL/6 mice. Arch. Biochem. Biophys. 527:95‐104. doi: 10.1016/j.abb.2012.03.018.
  Yamashita, Y., Ueda‐Wakagi, M., Sakamoto, M., Tachibana, N., Wanezaki, S., Kohno, M., and Ashida, H. 2015. β‐Conglycinin peptides improve glucose uptake through the AMPK signaling pathway in L6 myotubes. Food Sci. Technol. Res. 21:727‐732. doi: 10.3136/fstr.21.727.
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Key References
  Nishiumi and Ashida, 2007. See above.
  This article is the first report of the protocol described here.
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