Quantitative Analysis of Surface Expression of Membrane Proteins Using Cold‐Adapted Proteases

Faraz Ahmad1, Kai Kaila1, Peter Blaesse2

1 University of Helsinki, Helsinki, Finland, 2 Westfälische Wilhelms‐University Münster, Münster, Germany
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 3.11
DOI:  10.1002/0471140864.ps0311s73
Online Posting Date:  September, 2013
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Abstract

This unit presents an improved method for quantitative analysis of surface expression of membrane proteins utilizing a cold‐adapted trypsin. Preservation of the proteolytic activity of the enzyme at 0° to 4°C allows cleavage of surface‐expressed membrane proteins at temperatures at which trafficking of the mammalian plasmalemmal proteins is blocked. This provides an important advantage over established trypsin‐cleavage protocols since it can be applied to membrane proteins with a fast turnover rate of the membrane pool and a fast recycling rate. Compared to surface biotinylation, the method is less time consuming. Curr. Protoc. Protein Sci. 73:3.11.1‐3.11.12. © 2013 by JohnWiley & Sons, Inc.

Keywords: membrane protein; surface expression; trafficking; trypsin; biotinylation; BS3; surface analysis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Quantification of Surface Expression of Membrane Proteins in Slices from Animal Tissue
  • Basic Protocol 2: Quantification of Surface Expression of Membrane Proteins in Cell Culture Systems
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quantification of Surface Expression of Membrane Proteins in Slices from Animal Tissue

  Materials
  • Tissue for analysis
  • Physiological solution [e.g., artificial cerebrospinal fluid (ACSF); see recipe]
  • Trypsin from cod (e.g., Penzyme from Zymetech; see recipe and Commentary)
  • Protease inhibitor cocktail (e.g., Roche) or phenylmethylsulfonyl fluoride (PMSF; see recipe)
  • Homogenization buffer (e.g., RIPA buffer; see recipe)
  • Protein quantification kit (e.g., DC protein assay kit from Bio‐Rad)
  • Reagents for SDS‐PAGE, western blotting, and immunodetection
    • 3× SDS‐PAGE loading buffer (see recipe)
    • Pre‐stained protein molecular weight standard
    • SDS‐PAGE running buffer (see recipe)
    • Western blotting buffer (see recipe)
    • Phosphate‐buffered saline with Tween‐20 (PBST; see recipe)
    • Blocking buffer (see recipe)
    • ECL western blotting substrate (Pierce)
    • Appropriate primary and enzyme‐linked secondary antibodies (see Commentary)
  • Forceps
  • Homogenization pestles or sonicator
  • Nitrocellulose or polyvinylidene fluoride (PVDF) membrane
  • Image Reader for detection of immunoreactive signal (LAS 3000, Fujifilm)
  • Additional reagents and equipment for protein quantification (unit 3.4), SDS‐PAGE (unit 10.1), electrophoretic transfer (unit 10.7), and immunodetection (unit 10.10)

Basic Protocol 2: Quantification of Surface Expression of Membrane Proteins in Cell Culture Systems

  Materials
  • Cells expressing the protein of interest
  • Phosphate‐buffered saline (1× PBS), pH 7.4 ( appendix 2E)
  • Trypsin from cod (e.g., Penzyme from Zymetech; see recipe and Commentary)
  • Protease inhibitor cocktail (e.g., Roche) or phenylmethylsulfonyl fluoride (PMSF) (see recipe)
  • Homogenization buffer (e.g., RIPA buffer; see recipe)
  • Cell culture dishes, e.g., 35‐mm
  • Cell scraper
  • Additional reagents and equipment for protein quantification ( protocol 1 and unit 3.4), SDS‐PAGE ( protocol 1 and unit 10.1), electrophoretic transfer ( protocol 1 and unit 10.7), and immunodetection ( protocol 1 and unit 10.10)
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Figures

Videos

Literature Cited

Literature Cited
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