Identification of Protein Interactions by Far Western Analysis

Diane G. Edmondson1, Sharon Y. Roth1

1 M.D. Anderson Cancer Center, Houston, Texas
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.2
DOI:  10.1002/0471143030.cb1702s07
Online Posting Date:  May, 2001
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Abstract

Far Western blotting is a method for detecting protein‐protein interactions. This technique utilizes a nonantibody protein probe to detect interacting proteins immobilized on a membrane support. Proteins to be assayed can be prepared by multiple techniques and detected by several labeling schemes.

     
 
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Table of Contents

  • Basic Protocol 1: Far Western Analysis of a Protein Mixture
  • Alternate Protocol 1: Detecting Interacting Proteins by Immunoblotting
  • Alternate Protocol 2: Using Peptides to Identify Specific Interacting Sequences in a Far Western Blot
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Far Western Analysis of a Protein Mixture

  Materials
  • Samples to be analyzed
  • 1× SDS sample buffer ( appendix 2A)
  • Ponceau S staining solution (see recipe)
  • Blocking buffer I: 0.05% (w/v) Tween 20 in 1× PBS (see recipe for PBS); prepare fresh
  • Blocking buffer II: dissolve 1 g bovine serum albumin (BSA; fraction V) in 100 ml 1× PBS (see recipe for PBS); prepare fresh
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.9
  • cDNA encoding protein of interest cloned into an in vitro expression vector
  • In vitro transcription/translation kit (Promega)
  • 10 mCi/ml 35S‐methionine (1000 Ci/mmol)
  • Probe purification buffer (see recipe)
  • Probe dilution buffer ( recipe)
  • Polyvinyldifluoridine (PVDF) or nitrocellulose membrane for protein transfer
  • Microfiltration centrifuge columns (e.g., Gelman Nanosep, Pall Filtron, or Millipore Microcon)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), electrophoretic transfer of proteins to a support membrane (unit 6.2), in vitro translation (unit 11.2), and autoradiography (unit 6.3)
NOTE: Always handle support membranes with gloves or membrane forceps.

Alternate Protocol 1: Detecting Interacting Proteins by Immunoblotting

  • Recombinant protein or unlabeled in vitro translated–protein for probe
  • 5% (w/v) non‐fat instant dry milk in 1× TBST (see recipe for TBST)
  • Primary antibody specific for protein probe
  • TBST (see recipe)
  • Alkaline phosphatase (AP)–conjugated secondary antibody against Ig of species from which specific antibody was obtained
  • Alkaline phosphatase buffer (see recipe)
  • Developing solution (see recipe)
  • 100 mM EDTA, pH 8.0 ( appendix 2A)

Alternate Protocol 2: Using Peptides to Identify Specific Interacting Sequences in a Far Western Blot

  • Peptides
  • 0.4% Tween 20/PBS (see recipe)
  • India ink solution (see recipe)
  • Slot or dot blot apparatus (e.g., Bio‐Rad Bio‐Dot SF or Schleicher & Schuell Minifold II)
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Figures

Videos

Literature Cited

Literature Cited
   Chaudhary, J., Cupp, A.S., and Skinner, M.K. 1997. Role of basic‐helix‐loop‐helix transcription factors in Sertoli cell differentiation: Identification of an E‐box response element in the transferrin promoter. Endocrinology 138: 667‐ 675.
   Edmondson, D.G., Smith, M.M., and Roth, S.Y. 1996. Repression domain of the yeast global repressor Tup1 interacts directly with histones H3 and H4. Genes & Dev. 10: 1247‐ 1259.
   Fischer, N., Kremmer, E., Lautscham, G., Mueller‐ Lantzsch, N., and Grasser, F. A. 1997. Epstein‐Barr virus nuclear antigen 1 forms a complex with the nuclear transporter karyopherin alpha2. J. Biol. Chem. 272: 3999‐ 4005.
   Grasser, F. A., Sauder, C., Haiss, P., Hille, A., Konig, S., Gottel, S., Kremmer, E., Leinenbach, H. P., Zeppezauer, M., and Mueller‐ Lantzsch, N. 1993. Immunological detection of proteins associated with the Epstein‐Barr virus nuclear antigen 2A. Virology 195: 550‐ 560.
   Grulich‐ Henn, J., Spiess, S., Heinrich, U., Schonberg, D., and Bettendorf, M. 1998. Ligand blot analysis of insulin‐like growth factor‐binding proteins using biotinylated insulin‐like growth factor‐I. Horm. Res. 49: 1‐ 7.
   Hsiao, P. W., and Chang, C. 1999. Isolation and characterization of ARA160 as the first androgen receptor N‐terminal‐associated coactivator in human prostate cells. J. Biol. Chem. 274: 22373‐ 22379.
   Kimball, S.R., Heinzinger, N.K., Horetsky, R.L., and Jefferson, L.S., 1998. Identification of interprotein interactions between the subunits of eukaryotic initiation factors eIF2 and eIF2B. J. Biol.Chem. 273: 3039‐ 3044.
   Kleinschmidt, J.A., and Seiter, A. 1988. Identification of domains involved in nuclear uptake and histone binding of protein N1 of Xenopus laevis. EMBO J. 7:1605‐1614.
   Kouklis, P. D., Hutton, E., and Fuchs, E. 1994. Making a connection: Direct binding between keratin intermediate filaments and desmosomal proteins. J. Cell Biol. 127: 1049‐ 1060.
   Luna, E.J. 1996. Biotinylation of proteins in solution and on cell surfaces. In Current Protocols in Protein Science. ( J.E. Coligan, B.M. Dunn, H.L. Ploegh, D.W. Speicher, and P.T. Wingfield, eds.) pp. 3.6.1 ‐ 3.6.15. John Wiley & Sons, New York
   Palaparti, A., Baratz, A., and Stifani, S. 1997. The Groucho/transducin‐like enhancer of split transcriptional repressors interact with the genetically defined amino‐terminal silencing domain of histone H3. J. Biol. Chem. 272: 26604‐ 26610.
   Schumacher, T.N.M. and Tsomides, T.I. 1995. In vitro radiolabeling of peptides and proteins. In Current Protocols in Protein Science ( J.E. Coligan, B.M. Dunn, H.L. Ploegh, D.W. Speicher, and P.T. Wingfield. eds.) pp. 3.3.1‐ 3.3.19. John Wiley & Sons, New York.
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