Pulse‐Chase Analysis for Studying Protein Synthesis and Maturation

Susanne Fritzsche1, Sebastian Springer1

1 Department of Life Sciences and Chemistry, Jacobs University Bremen, Bremen
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 30.3
DOI:  10.1002/0471140864.ps3003s78
Online Posting Date:  November, 2014
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Abstract

Pulse‐chase analysis is a well‐established and highly adaptable tool for studying the life cycle of endogenous proteins, including their synthesis, folding, subunit assembly, intracellular transport, post‐translational processing, and degradation. This unit describes the performance and analysis of a radiolabel pulse‐chase experiment for following the folding and cell surface trafficking of a trimeric murine MHC class I glycoprotein. In particular, the unit focuses on the precise timing of pulse‐chase experiments to evaluate early/short‐time events in protein maturation in both suspended and strictly adherent cell lines. The advantages and limitations of radiolabel pulse‐chase experiments are discussed, and a comprehensive section for troubleshooting is provided. Further, ways to quantitatively represent pulse‐chase results are described, and feasible interpretations on protein maturation are suggested. The protocols can be adapted to investigate a variety of proteins that may mature in very different ways. © 2014 by John Wiley & Sons, Inc.

Keywords: metabolic labeling; protein folding; protein transport; secretory pathway; glycan processing; immunoprecipitation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Pulse‐Chase Analysis of Suspended Cell Lines
  • Alternate Protocol 1: Pulse‐Chase Analysis of Adherent Cell Lines
  • Support Protocol 1: Preparation of Antibody‐Conjugated Protein A–Agarose Beads
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Pulse‐Chase Analysis of Suspended Cell Lines

  Materials
  • K41 cells (Nakamura et al., ) in logarithmic growth
  • PBS, room temperature and 4°C
  • Starvation medium (see recipe), 37°C
  • Culture medium (see recipe), 4°C
  • [35S]cysteine/methionine (EasyTag EXPRESS35S protein labeling mix, Perkin Elmer; store at −80°C)
  • 20 mg/ml unlabeled methionine (tissue culture grade, sterile‐filtered, store at −80°C)
  • 100 mg/ml unlabeled cysteine (tissue culture grade, sterile‐filtered, store at −80°C)
  • Native lysis buffer (NLB, see recipe)
  • 500 mM iodoacetamide (IAA) in acetone (store at −20°C)
  • 500 mM phenylmethylsulfonylfluoride (PMSF) in dimethyl sulfoxide (DMSO) (store at −20°C)
  • Protein A (e.g., insoluble protein A from Staphylococcus aureus; Sigma‐Aldrich)
  • Antibody‐bound protein A–agarose beads (see protocol 3Support Protocol)
  • Conformation‐dependent MHC class I antibody Y3 (Hämmerling et al., )
  • Wash buffer (see recipe)
  • 10× denaturation buffer (see recipe)
  • 10× endoglycosidase reaction buffer (see recipe)
  • 20% (w/v) Triton X‐100 (store at 4°C)
  • Endoglycosidase F1 or H (EndoF1 or EndoH; e.g., New England Biolabs, Sigma‐Aldrich)
  • 6× SDS‐PAGE sample buffer (see recipe)
  • Radioactive marker, e.g., [methyl‐14C]methylated protein molecular weight markers (Perkin Elmer; stored at −80°C)
  • 50‐ml conical centrifuge tubes
  • Cell culture incubator (5% CO 2, 37°C) approved for radioactive work, with optional rotator
  • 1.5‐ml microcentrifuge tubes
  • 27‐G needle (optional)
  • 95° and 37°C heat block or thermoshaker
  • Quantitation software
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1), gel drying (unit 10.1), blotting (unit 10.7), and autoradiography (unit 10.11)

Alternate Protocol 1: Pulse‐Chase Analysis of Adherent Cell Lines

  Additional Materials
  • 10‐cm tissue culture dishes
  • Cell scraper

Support Protocol 1: Preparation of Antibody‐Conjugated Protein A–Agarose Beads

  Materials
  • Protein A–agarose beads (Calbiochem, Merck Millipore)
  • Wash buffer (see recipe)
  • 50‐ml conical tube
  • 1.5‐ml microcentrifuge tubes
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Figures

Videos

Literature Cited

Literature Cited
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