Analysis of Disulfide Bond Formation

Ineke Braakman1, Daniel N. Hebert2

1 University of Amsterdam, Academic Medical Center, 2 Yale University School of Medicine, New Haven, Connecticut
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.1
DOI:  10.1002/0471140864.ps1401s03
Online Posting Date:  May, 2001
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Abstract

In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein is then chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described. A support protocol is provided for analysis of disulfide bonds in the immunoprecipitates by SDSā€PAGE with and without prior reduction. The difference in mobility observed between the gels with unreduced and reduced samples is due to disulfide bonds in the unreduced protein.

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of Disulfide Bond Formation in Intact Monolayer Cells
  • Alternate Protocol 1: Analysis of Disulfide Bond Formation in Cells in Suspension
  • Alternate Protocol 2: Analysis of Post‐Translational Disulfide Bond Formation in Intact Cells
  • Basic Protocol 2: Analysis of Disulfide Bond Formation in Rough Endoplasmic Reticulum–Derived Microsomes
  • Alternate Protocol 3: Analysis of Post‐Translational Disulfide Bond Formation in Rough Endoplasmic Reticulum–Derived Microsomes
  • Support Protocol 1: Immunoprecipitation of Lysates
  • Support Protocol 2: Nonreducing and Reducing SDS‐PAGE
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Analysis of Disulfide Bond Formation in Intact Monolayer Cells

  Materials
  • Adherent cells
  • Tissue culture medium containing methionine, 37°C
  • recipeWash buffer (see recipe), 37°C
  • recipeDepletion medium (see recipe), 37°C
  • recipeLabeling medium (containing 125 to 250 µCi/ml [35S]methionine; see recipe), 37°C
  • recipeChase medium (see recipe), 37°C
  • recipeStop buffer (see recipe), 0°C
  • recipeLysis buffer (see recipe), 0°C
  • 60‐mm tissue culture dishes, sterile
  • 37°C humidified 5% CO 2 incubator
  • 37°C water bath with rack or insert to hold tissue culture dishes (e.g., Unwire racks for 15‐ and 50‐ml tubes, Nalge)
  • Aspiration flask for radioactive waste
  • Flat, wide ice pan with fitted metal plate (e.g., VWR Scientific)
  • Cell scraper
  • Additional reagents and equipment for immunoprecipitation (see protocol 6) and nonreducing and reducing SDS‐PAGE (see protocol 7)
NOTE: The volumes described here are for a 60‐mm dish of cells. Volumes must be adjusted, based on the surface area of the dish, for other sizes.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Analysis of Disulfide Bond Formation in Cells in Suspension

  • Culture of suspension cells >1000
  • 10 mCi/ml [35S]methionine (>1000 Ci/mmol; Amersham)
  • recipe2× lysis buffer (see recipe)
  • recipeConcentrated chase medium (see recipe)
  • 50‐ml polystyrene tube with cap, sterile
  • Beckman GPR cell centrifuge or equivalent
  • Additional reagents and equipment for immunoprecipitation (see protocol 6) and nonreducing SDS‐PAGE (see protocol 7)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: Keep cells in suspension during incubations by gently swirling the tube at regular intervals.

Alternate Protocol 2: Analysis of Post‐Translational Disulfide Bond Formation in Intact Cells

  Materials
  • 1 equivalent/µl nuclease‐treated dog pancreas microsomes (Promega)
  • Rabbit reticulocyte lysate treated with ATP‐regenerating system and nucleases (Promega)
  • 1 mM amino acid mixture lacking methionine (Promega)
  • 10 mCi/ml [35S]methionine (1000 Ci/mmol, Amersham)
  • 100 mM dithiothreitol (DTT)
  • RNase‐free H 2O (e.g., recipeDEPC‐treated; see recipe)
  • 23 U/ml RNase inhibitor (e.g., RNasin, Promega)
  • 1 µg/µl mRNA for the protein of interest
  • 100 mM oxidized glutathione (GSSG; appendix 3A), titrated to neutrality with KOH
  • recipe120 mM N‐ethylmaleimide (NEM) in 100% ethanol (prepare from 1 M stock; see recipe)
  • recipeLysis buffer (see recipe)
  • recipe2× SDS sample buffer ( unit 10.1; optional)
  • 27°C water bath
  • 1.5‐ml microcentrifuge tubes, RNase‐free
  • Ice bath
  • Additional reagents and equipment for immunoprecipitation (see protocol 6) and nonreducing SDS‐PAGE (see protocol 7)
NOTE: The major source of failure in the in vitro translation/translocation of proteins is contamination with ribonucleases (RNases) that degrade mRNA. To avoid contamination, water and salt solutions should be treated with diethylpyrocarbonate (DEPC) to chemically inactivate RNases; glass and plasticware should be treated with DEPC‐treated water or otherwise treated to remove RNase activity (see recipe for recipeDEPC treatment). Freshly opened plasticware that has not been touched by unprotected hands is also acceptable. It is also helpful to keep a set of solutions for RNA work alone to ensure that “dirty” pipets do not contaminate them. In addition, gloves should be worn at all times.

Basic Protocol 2: Analysis of Disulfide Bond Formation in Rough Endoplasmic Reticulum–Derived Microsomes

  Materials
  • 10% (w/v) killed, fixed Staphylococcus aureus cells (Zymed)
  • Antibody against protein of interest
  • Lysate from pulse‐chase labeled cells or microsomes (see protocol 1 or protocol 42 or protocol 2, protocol 32, or protocol 53)
  • recipeImmunoprecipitation wash buffer (see recipe), 37°C
  • recipeTE buffer, pH 6.8 (see recipe)
  • recipe2× nonreducing sample buffer (see recipe)
  • 200 mM dithiothreitol (DTT)
  • Rotator
  • Additional reagents and equipment for nonreducing and reducing SDS‐PAGE (see protocol 7)

Alternate Protocol 3: Analysis of Post‐Translational Disulfide Bond Formation in Rough Endoplasmic Reticulum–Derived Microsomes

  Materials
  • Samples in 1× sample buffer (see protocol 6)
  • recipe2× nonreducing sample buffer (see recipe)
  • PBS ( appendix 2E)/30% (v/v) methanol
  • 1.5 M salicylate/30% (v/v) methanol
  • pH paper
  • Whatman 3MM filter paper
  • Additional reagents and equipment for SDS‐PAGE minigel with Laemmli buffers (unit 10.1) and Coomassie blue staining and destaining (unit 10.5)
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Figures

Videos

Literature Cited

Literature Cited
   Blobel, G. and Dobberstein, B. 1975. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J. Cell Biol. 67:852‐862.
   Braakman, I., Hoover‐Litty, H., Wagner, K.R., and Helenius, A. 1991. Folding of influenza hemagglutinin in the endoplasmic reticulum. J. Cell Biol. 114:401‐411.
   Braakman, I., Helenius, J., and Helenius, A. 1992a. Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum. EMBO J. 11:1717‐1722.
   Braakman, I., Helenius, J., and Helenius, A. 1992b. Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum. Nature 356:260‐262.
   Bulleid, N.J. and Freedman, R. 1988. Defective cotranslational formation of disulphide bonds in protein disulphide‐isomerase‐deficient microsomes. Nature 335:649‐651.
   Creighton, T.E. 1986. Disulfide bonds as probes of protein folding pathways. Methods Enzymol. 131:83‐106.
   Hebert, D.N., Foellmer, B., and Helenius, A. 1995. Glucose trimming and reglycosylation determine glycoprotein association with calnexin in the endoplasmic reticulum. Cell 81:425‐433.
   Marquardt, T., Hebert, D.N., and Helenius, A. 1993. Post‐translational folding of Influenza hemagglutinin in isolated endoplasmic reticulum–derived microsomes. J. Biol. Chem. 268:19618‐19625.
   Nicchitta, C.V. and Blobel, G. 1993. Lumenal proteins of the mammalian endoplasmic reticulum are required to complete protein translocation. Cell 73:989‐998.
   Scheele, G. and Jacoby, R. 1982. Conformational changes associated with proteolytic processing of presecretory proteins allow glutathione‐catalyzed formation of native disulfide bonds. J. Biol. Chem. 257:12277‐12282.
Key References
   Braakman et al., 1991. See above.
  Describes the protocol in intact cells, results with influenza hemagglutinin, and considerations for ultra‐short pulse times.
   Hebert et al., 1995. See above.
  Describes the protocol in microsomes.
   Marquardt et al., 1993. See above.
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