Detection of Stress Proteins as Biomarkers of Oxidative Stress

Linda L. Agnew1, Kenneth Watson1

1 University of New England, Armidale
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.8
DOI:  10.1002/0471140856.tx1708s28
Online Posting Date:  June, 2006
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Abstract

Heat shock or stress proteins are constitutively expressed redox‐sensitive proteins, the synthesis of which is induced in almost all organisms exposed to a range of stressors, including oxidative stress, heat shock, free radicals, UV radiation, and heavy metals. This unit details three methods, with supporting protocols, for their measurement in peripheral blood mononuclear cells, plasma, and serum. These are [35S]methionine protein labeling, immunoblotting, and ELISA.

Keywords: heat shock proteins; oxidative stress; immunoblot; ELISA

     
 
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Table of Contents

  • Basic Protocol 1: Detection of Stress Proteins by SDS‐Page and Autoradiography
  • Basic Protocol 2: Detecting Stress Proteins by Immunoblotting
  • Basic Protocol 3: Detection of Stress Proteins Using ELISA
  • Support Protocol 1: Ficoll‐Paque Isolation of PBMC from Whole Blood
  • Support Protocol 2: Inducing Stress Proteins by Heat Shock
  • Support Protocol 3: Inducing Stress Proteins by Oxidative Stress
  • Support Protocol 4: Protein Extraction
  • Support Protocol 5: Estimation of Protein Concentration
  • Support Protocol 6: Preparation of Protein Samples for Electrophoresis
  • Support Protocol 7: Densitometry of Gels
  • Support Protocol 8: Stripping of Immunoblot Membranes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of Stress Proteins by SDS‐Page and Autoradiography

  Materials
  • 70% ethanol
  • 30% (w/v) acrylamide/0.8% (w/v) bisacrylamide (see recipe)
  • 1.5 M Tris·Cl, pH 8.8 ( appendix 2A)
  • TEMED
  • 10% (w/v) sodium dodecyl sulfate (SDS)
  • 10% (w/v) ammonium persulfate (prepare fresh)
  • 1 M Tris·Cl, pH 6.8 ( appendix 2A)
  • 1× running buffer (see recipe for 10× stock)
  • Prestained SDS standard molecular weight marker (e.g., Bio‐Rad)
  • Protein samples extracted from PBMC that have been exposed to stressor (see Support Protocols protocol 74, protocol 85, and protocol 96)
  • Gel fixative (see recipe)
  • Kodak developer and fixer or equivalent
  • Glass gel‐casting plates (20 × 22–cm) and 1‐mm spacers (or use equipment supplied with Bio‐Rad Protean II system)
  • Syringes
  • Filter paper
  • Plastic well combs, 15‐ or 25‐well
  • Gel electrophoresis equipment (e.g., Bio‐Rad Protean II xi Cell)
  • Thermocirculator suitable for use at 10°C
  • Gel‐loading pipet tips
  • Power supply with 200‐mA capability
  • Platform rocker
  • Blotting paper
  • Cellophane
  • Pyrex dish for fixation, large enough to fit gel
  • Kodak Biomax MR film
  • Film cassette
  • Additional reagents and equipment for polyacrylamide gel electrophoresis ( appendix 3F) and autoradiography ( appendix 3D)

Basic Protocol 2: Detecting Stress Proteins by Immunoblotting

  Materials
  • Protein samples extracted from PBMCs (Support Protocols protocol 74, protocol 85, and protocol 96), concentration determined by Coomassie protein assay ( protocol 8)
  • Prestained molecular weight marker (e.g., Bio‐Rad Kaleidoscope marker)
  • Precast 10% (w/v) polyacrylamide electrophoresis gels (e.g., Gradipore)
  • 1× running buffer (see recipe for 10× stock solution)
  • Transfer buffer (see recipe)
  • PBS‐Tween (PBS‐T; see recipe)
  • Incubation/blocking buffer (see recipe)
  • Primary antibody: anti‐hsp antibody of interest (e.g., anti‐hsp70; StressGen Biotechnologies)
  • ECL western blotting detection kit (Amersham)
  • Photographic developer and fixer (e.g., Kodak)
  • Anti‐β actin antibody (Sigma‐Aldrich)
  • Mini‐vertical gel unit (e.g., Gradipore)
  • Power supply
  • Wash containers: rectangular plastic trays of size appropriate to accommodate two gels
  • Template with same dimensions as gel for cutting filter paper and membranes
  • Whatman no. 41 filter paper
  • Hybond C Super nitrocellulose membrane (Amersham)
  • Electrophoresis Transfer Unit (e.g., NovaBlot; Amersham Biosciences) and power supply
  • Glass rod
  • Forceps
  • Scalpel
  • Plastic wash container with lid, of dimensions to accommodate membrane
  • Heat‐sealable plastic bags
  • Thermosealer
  • 15‐ml centrifuge tubes
  • Darkroom with red safelight
  • Hyperfilm ECL (Amersham)
  • Film cassette
  • Airtight container for storing membranes
  • Additional reagents and equipment for polyacrylamide gel electrophoresis ( appendix 3F), autoradiography (optional), densitometry ( protocol 10), and stripping of immunoblot membranes ( protocol 11)

Basic Protocol 3: Detection of Stress Proteins Using ELISA

  Materials
  • Hsp70 ELISA kit (StressGen Biotechnologies) containing:
    • hsp70 immunoassay plate
    • 5× hsp70 extraction reagent
    • Recombinant hsp70 standard
    • Sample diluent
    • 10× wash buffer
    • Anti‐hsp70 biotin conjugate
    • Anti‐hsp70 biotin conjugate diluent
    • Avidin‐HRP conjugate
    • Avidin‐HRP conjugate diluent
    • TMB substrate
    • Acid stop solution
    • Instruction manual
  • Protein samples extracted from PBMC ( protocol 7) or serum/plasma samples
  • Bleach
  • Adhesive plate covers
  • Platform rocker
  • 50‐ml conical centrifuge tubes
  • ELISA microplate reader

Support Protocol 1: Ficoll‐Paque Isolation of PBMC from Whole Blood

  Materials
  • Human blood donor
  • RPMI 1640 medium without methionine (ICN Biochemicals)
  • Ficoll‐Paque Plus (Amersham‐Pharmacia)
  • RPMI 1640 medium without methionine supplemented with 10% (w/v) fetal bovine serum (FBS)
  • 10‐ml lithium‐heparin peripheral blood collection Vacutainers
  • 50‐ml conical screw‐cap centrifuge tubes
  • 10‐ml syringes
  • Mixing cannulae, sterile (Indoplas; http://www.unomedical.com), to fit 10‐ml syringe
  • 50‐ml plastic, screw‐cap centrifuge tubes
  • Refrigerated centrifuge
  • Plastic 3.5‐ml transfer pipets, sterile

Support Protocol 2: Inducing Stress Proteins by Heat Shock

  Materials
  • Isolated PBMC (see protocol 4)
  • Trans[35S]methionine metabolic labeling reagent (≥70% L‐[35S]methionine, ≤15% L‐[35S]cysteine; specific activity >1000 Ci/mmol; ICN Biochemicals)
  • 37°C and 42.5°C water baths

Support Protocol 3: Inducing Stress Proteins by Oxidative Stress

  Materials
  • Isolated PBMC (see protocol 4)
  • Trans[35S]methionine metabolic labeling reagent (≥70% L‐[35S]methionine, ≤15% L‐[35S]cysteine, specific activity >1000 Ci/mmol; ICN Biochemicals)
  • Appropriate chemicals for induction of oxidative stress: e.g., hydrogen peroxide (Sigma‐Aldrich) or 2,2′‐azobis‐(2‐amidinopropane) dihydrochloride (AAPH; Wako Pure Chemicals)
  • 37° and (optionally, if heat shock in addition to oxidative stress is required) 42.5°C water baths

Support Protocol 4: Protein Extraction

  Materials
  • Radiolabeled stressed and control PBMC suspensions ( protocol 5 or protocol 6)
  • RPMI 1640 medium without methionine (ICN Biochemicals)
  • Protein extraction buffer (see recipe)
  • Centrifuge
  • Additional reagents and equipment for protein estimation assay ( protocol 8)

Support Protocol 5: Estimation of Protein Concentration

  Materials
  • PBMC protein extracts ( protocol 7)
  • Bovine serum albumin (BSA; Pierce)
  • Coomassie protein detection reagent (Pierce)
  • Plastic 2‐ml cuvettes
  • Spectrophotometer capable of reading absorbance at 595 nm

Support Protocol 6: Preparation of Protein Samples for Electrophoresis

  Materials
  • PBMC protein extracts ( protocol 7)
  • 2× Gel loading buffer (see recipe)
  • Boiling water bath

Support Protocol 7: Densitometry of Gels

  Materials
  • Samples run on a gel and autoradiographed or immunoblotted ( protocol 1 or protocol 2)
  • Densitometric gel documentation and analysis system (e.g., UVP)
  • One‐dimensional SDS‐PAGE densitometry software package (e.g., Phoretix 1D Advanced, Nonlinear Dynamics; http://www.nonlinear.com)

Support Protocol 8: Stripping of Immunoblot Membranes

  Materials
  • Previously probed immunoblot membrane ( protocol 2)
  • Membrane stripping buffer (see recipe)
  • PBS‐Tween (PBS‐T; see recipe)
  • Incubation/blocking buffer (see recipe)
  • 50°C incubator or heat block
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Figures

Videos

Literature Cited

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Key References
   Laemmli, 1970. See above.
  Describes the procedure for polyacrylamide gel electrophoresis for separation of proteins. A modification of this method is described in .
   Liao et al., 2000. See above.
  Describes the use of β‐actin as an internal control for immunoblots. A modification of this method is described in Support Protocols 6 and 7.
   Peng et al., 2000. See above.
  Describes typical results obtained in PBMC that have been heat shocked and oxidatively stressed with AAPH. Stressed induced synthesis of hsp 32 (HO‐1) and hsps are analyzed by 35S‐methionine protein labeling, SDS‐PAGE and by immunoblot.
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