Thioredoxin Redox Western Analysis

Young‐Mi Go1, Dean P. Jones1

1 Emory University, Atlanta, Georgia
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.12
DOI:  10.1002/0471140856.tx1712s41
Online Posting Date:  August, 2009
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Abstract

Increasing evidence suggests that compartmentspecific changes are important in redox signaling and control. Examination of thiol/disulfide redox changes in thioredoxin (Trx) family members including Trx1 in cytoplasm and nucleus and Trx2 in mitochondria should aid in the understanding of compartmentalized redox signaling mechanisms. Methods to quantify redox states of both Trx1 and Trx2 by redox western analysis and to further calculate redox potential using the Nernst equation are described in this unit. The procedures to measure redox states of Trx1 and Trx2 consist of three parts, derivatization, redox western blotting, and calculation of redox potentials. Derivatization of proteins with thiol‐reactive reagents prior to redox western blotting prevents artifactual oxidation of protein thiols and allows separation of the reduced forms from oxidized forms of the protein. Consequently, the redox western analysis of Trx provides a convenient tool to estimate the redox state of different compartments and improve the understanding of redox signaling. Curr. Protoc. Toxicol. 41:17.12.1‐17.12.12. © 2009 by John Wiley & Sons, Inc.

Keywords: redox potential; redox state; thiol‐disulfide; cysteine

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Trx1 Redox Potential
  • Alternate Protocol 1: Measurement of Trx2 Redox Potential
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of Trx1 Redox Potential

  Materials
  • G‐lysis buffer with 50 mM IAA, pH 8.3 (see recipe), cold
  • Cultured cells (1–2 × 106) growing in a 6‐well plate or tissue (10 to 100 mg wet weight)
  • Human cell lines (e.g., HeLa cells) or purified Trx1 protein
  • 0.5 M dithiothreitol (DTT; see recipe)
  • 1.0 M hydrogen peroxide (H 2O 2; see recipe)
  • 1× phosphate‐buffered saline (PBS; appendix 2A), cold
  • Iodoacetic acid (IAA, Sigma‐Aldrich)
  • G‐lysis buffer with 100 mM IAA, pH 8.3 (see recipe for G‐lysis buffer)
  • 15% native, non‐reducing polyacrylamide separating gel (see recipe)
  • 6% native, non‐reducing polyacrylamide stacking gel (see recipe)
  • 5× native and non‐reducing sample loading buffer (see recipe)
  • 1× native running buffer (see recipe)
  • 1× transfer buffer (see recipe)
  • Odyssey blocking buffer (LI‐COR Biosciences)
  • PBST (0.1 % Tween‐20 in PBS)
  • Primary antibody against Trx1
  • Infrared dye (680 nm or 800 nm)–labeled secondary antibodies
  • Cell scraper
  • 1.5‐ml microcentrifuge tubes
  • 37°C incubator in the dark
  • Scissors or Dounce homogenizer
  • G‐25 microspin columns (GE Healthcare)
  • Gel apparatus and power source
  • Nitrocellulose membrane (0.2‐µm)
  • Platform shaker, room temperature
  • Odyssey scanner and software (LI‐COR Biosciences)

Alternate Protocol 1: Measurement of Trx2 Redox Potential

  • Cultured cells (1–2 × 106) in 35‐mm dish or one well of 6‐well plate or tissue (10 to 100 mg)
  • 10% trichloroacetic acid (TCA; see recipe)
  • 100% acetone
  • Lysis/derivatization buffer (see recipe)
  • 15% polyacrylamide separating gel (see recipe)
  • 6% polyacrylamide stacking gel (see recipe)
  • 5× non‐reducing sample loading buffer (see recipe)
  • 1× running buffer (see recipe)
  • Primary antibody against Trx2
  • Vortexer
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Figures

Videos

Literature Cited

   Chen, Y., Cai, J., Murphy, T.J., and Jones, D.P. 2002. Overexpressed human mitochondrial thioredoxin confers resistance to oxidant‐induced apoptosis in human osteosarcoma cells. J. Biol. Chem. 277:33242‐33248.
   Chen, Y., Cai, J., and Jones, D.P. 2006. Mitochondrial thioredoxin in regulation of oxidant‐induced cell death. FEBS Lett. 580:6596‐6602.
   Fernando, M.R., Nanri, H., Yoshitake, S., Nagata‐Kuno, K., and Minakami, S. 1992. Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells. Eur. J. Biochem. 209:917‐922.
   Go, Y.M. and Jones, D.P. 2008. Redox compartmentalization in eukaryotic cells. Biochim. Biophys. Acta 1780:1273‐1290.
   Go, Y.M., Ziegler, T.R., Johnson, J.M., Gu, L., Hansen, J.M., and Jones, D.P. 2007. Selective protection of nuclear thioredoxin‐1 and glutathione redox systems against oxidation during glucose and glutamine deficiency in human colonic epithelial cells. Free Radic. Biol. Med. 42:363‐370.
   Gutscher, M., Pauleau, A.L., Marty, L., Brach, T., Wabnitz, G.H., Samstag, Y., Meyer, A.J., and Dick, T.P. 2008. Real‐time imaging of the intracellular glutathione redox potential. Nat. Methods 5:553‐559.
   Halvey, P.J., Watson, W.H., Hansen, J.M., Go, Y.M., Samali, A., and Jones, D.P. 2005. Compartmental oxidation of thiol‐disulphide redox couples during epidermal growth factor signaling. Biochem. J. 386:215‐219.
   Hansen, J.M., Zhang, H., and Jones, D.P. 2006. Mitochondrial thioredoxin‐2 has a key role in determining tumor necrosis factor‐alpha‐induced reactive oxygen species generation, NF‐kappaB activation, and apoptosis. Toxicol. Sci. 91:643‐650.
   Hirota, K., Matsui, M., Iwata, S., Nishiyama, A., Mori, K., and Yodoi, J. 1997. AP‐1 transcriptional activity is regulated by a direct association between thioredoxin and Ref‐1. Proc. Natl. Acad. Sci. U.S.A. 94:3633‐3638.
   Holmgren, A. and Fagerstedt, M. 1982. The in vivo distribution of oxidized and reduced thioredoxin in Escherichia coli. J. Biol. Chem. 257:6926‐6930.
   Jones, D.P. 2008. Radical‐free biology of oxidative stress. Amer. J. Physiol. 295:C849‐C868.
   Kim, J.R., Lee, S.M., Cho, S.H., Kim, J.H., Kim, B.H., Kwon, J., Choi, C.Y., Kim, Y.D., and Lee, S.R. 2004. Oxidation of thioredoxin reductase in HeLa cells stimulated with tumor necrosis factor‐alpha. FEBS Lett. 567:189‐196.
   Kim, Y.J., Lee, W.S., Ip, C., Chae, H.Z., Park, E.M., and Park, Y.M. 2006. Prx1 suppresses radiation‐induced c‐Jun NH2‐terminal kinase signaling in lung cancer cells through interaction with the glutathione S‐transferase Pi/c‐Jun NH2‐terminal kinase complex. Cancer Res. 66:7136‐7142.
   Leichert, L.I., Gehrke, F., Gudiseva, H.V., Blackwell, T., Ilbert, M., Walker, A.K., Strahler, J.R., Andrews, P.C., and Jakob, U. 2008. Quantifying changes in the thiol redox proteome upon oxidative stress in vivo. Proc. Natl. Acad. Sci. U.S.A. 105:8197‐8205.
   Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata, M., Miyazono, K., and Ichijo, H. 1998. Mammalian thioredoxin is a direct inhibitor of apoptosis signal‐regulating kinase (ASK) 1. EMBO J. 17:2596‐2606.
   Watson, W.H., Pohl, J., Montford, W.R., Stuchlik, O., Reed, M.S., Powis, G., and Jones, D.P. 2003. Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif. J. Biol. Chem. 278:33408‐33415.
Key References
   Watson et al., 2003. See above.
  This reference describes redox characteristics of cysteine residues in Trx1 and determination of the midpoint potential of Trx1 using redox western blotting technique.
   Halvey et al., 2005. See above.
  This reference describes physiological stimuli, EGF‐induced compartmentalized oxidation, which was determined by measuring Trx1, Trx2, and glutathione redox states.
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