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

  • 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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Literature Cited

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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.
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  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.
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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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