Two‐Dimensional Blue Native Polyacrylamide Gel Electrophoresis

Wolfgang W.A. Schamel1

1 Max Planck‐Institut fµr Immunbiologie und Universität Freiburg, Biologie III, Freiburg, Germany
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 6.10
DOI:  10.1002/0471143030.cb0610s38
Online Posting Date:  March, 2008
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Abstract

Multiprotein complexes play crucial roles in nearly all cell biological processes. Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE) is a powerful method to study these complexes. It is a native protein separation method that relies on the dye Coomassie blue to confer negative charge for separation. It has a higher resolution than gel filtration or sucrose density ultracentrifugation and can be used for protein complexes from 10 kDa to 10 MDa. If a second‐dimension SDS‐PAGE is applied (two‐dimensional BN/SDS‐PAGE), the size, subunit composition, and relative abundance of the different multiprotein complexes can be studied. In recent years, there has been a large increase in the number of publications where BN‐PAGE was used to study protein‐protein interactions. Here, we give detailed protocols for the separation of multiprotein complexes by two‐dimensional BN/SDS‐PAGE and for a related technique to determine the stoichiometry of these complexes. Curr. Protoc. Cell Biol. 38:6.10.1‐6.10.21. © 2008 by John Wiley & Sons, Inc.

Keywords: multiprotein complex; native; gel electrophoresis; two‐dimensional; Coomassie blue; protein‐protein interaction

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: First‐Dimension Blue Native Electrophoresis
  • Support Protocol 1: Preparation of Cell Lysates for Blue Native Gel Electrophoresis
  • Basic Protocol 2: Second‐Dimension Denaturing Electrophoresis
  • Alternate Protocol 1: Denaturing Transfer of the Proteins from the First‐Dimension BN‐PAGE Gel to a Membrane for Immunoblotting
  • Alternate Protocol 2: Native Transfer of the Proteins from the First‐Dimension BN‐PAGE Gel to a Membrane for Immunoblotting
  • Basic Protocol 3: Native Antibody‐Based Mobility Shift (NAMOS) Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: First‐Dimension Blue Native Electrophoresis

  Materials
  • Low‐percentage BN separating gel solution (see recipe)
  • High‐percentage BN separating gel solution (see recipe)
  • Isobutyl alcohol
  • 3× BN gel buffer (see recipe)
  • BN stacking gel solution (see recipe)
  • 100× pervanadate solution (optional, if phosphorylation must be preserved; see recipe)
  • Sample: dialyzed cell lysate ( protocol 2), tissue homogenate, purified protein complex
  • Marker mixes 1 and 2 (see recipe)
  • BN anode buffer (see recipe)
  • BN cathode buffer (with 0.02% w/v Coomassie blue; see recipe)
  • BN cathode buffer (with low, 0.002% w/v Coomassie; see recipe)
  • Gel electrophoresis apparatus (see unit 6.1)
  • Gradient mixer (self‐made or, e.g., from BioRad; Fig. )
  • Peristaltic pump (Fig. )
  • Power supply
  • Additional reagents and equipment for polyacrylamide gel electrophoresis (unit 6.1) and protein staining in gels (unit 6.6)

Support Protocol 1: Preparation of Cell Lysates for Blue Native Gel Electrophoresis

  Materials
  • Cell culture dish (10 to 15 cm) containing cells of interest (80% confluent)
  • Phosphate‐buffered saline (PBS; see recipe), ice cold
  • BN lysis buffer (see recipe)
  • BN dialysis buffer (see recipe)
  • Cell scrapers
  • 10‐ to 50‐ml centrifuge tubes
  • Refrigerated cell culture centrifuge (with adaptor cavities for microcentrifuge tubes in rotor accommodating 50‐ml tube)
  • Dialysis membranes, MWCO 10 to 50 kDa (boiled and kept at 4°C in 0.001 M EDTA)
  • 1‐ml reaction tubes (e.g., microcentrifuge tubes)
  • Beaker (100‐ml to 1‐liter, depending on sample size)

Basic Protocol 2: Second‐Dimension Denaturing Electrophoresis

  Materials
  • 2× SDS sample buffer (unit 6.1)
  • 1× SDS electrophoresis buffer (unit 6.1)
  • Lane from a BN‐PAGE gel ( protocol 1)
  • Thin adhesive tape (e.g., tesa tape; http://www.tesatape.com)
  • Platform shaker
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and two‐dimensional gel electrophoresis (unit 6.4)

Alternate Protocol 1: Denaturing Transfer of the Proteins from the First‐Dimension BN‐PAGE Gel to a Membrane for Immunoblotting

  Materials
  • First‐dimension BN‐PAGE gel ( protocol 1)
  • Phosphate buffered saline (PBS; see recipe for 10×) containing 1% (w/v) SDS
  • Denaturing BN transfer buffer (see recipe)
  • Destaining solution (see recipe)
  • Platform shaker
  • PVDF (polyvinylidene fluoride) membrane (see unit 6.2)
  • Additional reagents and equipment for immunoblotting (unit 6.2)

Alternate Protocol 2: Native Transfer of the Proteins from the First‐Dimension BN‐PAGE Gel to a Membrane for Immunoblotting

  Materials
  • First‐dimension BN‐PAGE gel ( protocol 1)
  • Native BN transfer buffer (see recipe)
  • Destaining solution (see recipe)
  • PVDF membrane (see unit 6.2)
  • Additional reagents and equipment for immunoblotting (unit 6.2)

Basic Protocol 3: Native Antibody‐Based Mobility Shift (NAMOS) Assay

  Materials
  • Monoclonal antibodies that react with the proteins of interest
  • BN dialysis buffer (see recipe)
  • Sample for BN‐PAGE
  • 1‐ml reaction tubes (e.g., microcentrifuge tubes)
  • Additional reagents and equipment for casting and running BN‐PAGE gels (see protocol 1) and analysis of Blue Native gels by immunoblotting ( protocol 4 or protocol 52)
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Figures

Videos

Literature Cited

   Camacho‐Carvajal, M.M., Wollscheid, B., Aebersold, R., Steimle, V., and Schamel, W.W. 2004. Two‐dimensional blue native/SDS gel electrophoresis of multi‐protein complexes from whole cellular lysates: A proteomics approach. Mol. Cell. Proteomics 3: 176‐182.
   Dudkina, N.V., Eubel, H., Keegstra, W., Boekema, E.J., and Braun, H.P. 2005. Structure of a mitochondrial supercomplex formed by respiratory‐chain complexes I and III. Proc. Natl. Acad. Sci. U.S.A. 102: 3225‐3229.
   Millar, A.H., Heazlewood, J.L., Kristensen, B.K., Braun, H.P., and Moller, I.M. 2005. The plant mitochondrial proteome. Trends Plant Sci. 10: 36‐43.
   Model, K., Meisinger, C., Prinz, T., Wiedemann, N., Truscott, K.N., Pfanner, N., and Ryan, M.T. 2001. Multistep assembly of the protein import channel of the mitochondrial outer membrane. Nat. Struct. Biol. 8: 361‐370.
   Poetsch, A., Neff, D., Seelert, H., Schägger, H., and Dencher, N.A. 2000. Dye removal, catalytic activity and 2D crystallization of chloroplast H(+)‐ATP synthase purified by blue native electrophoresis. Biochim. Biophys. Acta 1466: 339‐349.
   Rexroth, S., Meyer zu Tittingdorf, J.M., Krause, F., Dencher, N.A., and Seelert, H. 2003. Thylakoid membrane at altered metabolic state: Challenging the forgotten realms of the proteome. Electrophoresis 24: 2814‐2823.
   Schamel, W.W., Arechaga, I., Risueno, R.M., van Santen, H.M., Cabezas, P., Risco, C., Valpuesta, J.M., and Alarcon, B. 2005. Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response. J. Exp. Med. 202: 493‐503.
   Schägger, H. 1995. Quantification of oxidative phosphorylation enzymes after blue native electrophoresis and two‐dimensional resolution: Normal complex I protein amounts in Parkinson's disease conflict with reduced catalytic activities. Electrophoresis 16: 763‐770.
   Schägger, H. and von Jagow, G. 1991. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199: 223‐231.
   Schägger, H., Cramer, W.A., and von Jagow, G. 1994. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two‐dimensional native electrophoresis. Anal. Biochem. 217: 220‐230.
   Schägger, H., Bentlage, H., Ruitenbeek, W., Pfeiffer, K., Rotter, S., Rother, C., Bottcher‐Purkl, A., and Lodemann, E. 1996. Electrophoretic separation of multiprotein complexes from blood platelets and cell lines: Technique for the analysis of diseases with defects in oxidative phosphorylation. Electrophoresis 17: 709‐714.
   Swamy, M., Minguet, S., Siegers, G.M., Alarcon, B., and Schamel, W.W. 2007. A native antibody‐based mobility‐shift technique (NAMOS‐assay) to determine the stoichiometry of multiprotein complexes. J. Immunol. Methods 324: 74‐83.
Key References
   Camacho‐Carvajal et al., 2004. See above.
  Describes the separation of cellular lysates by BN‐PAGE.
   Schägger and von Jagow, 1991. See above.
  Describes, for first time, BN‐PAGE and second‐dimension BN/SDS‐PAGE using solubilized mitochondria.
   Swamy et al., 2007. See above.
  Details the NAMOS assay with an extensive discussion of anticipated results.
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