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Identification of Protein Interactions by Far Western Analysis

Diane G. Edmondson¹, Sharon Y. Roth¹

¹M.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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Unit Introduction
Basic Protocol: 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: 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

Additional Materials (also see Basic Protocol)

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

Additional Materials (also see Basic Protocol)

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

Figure 17.2.1

Far western of blotted SDS-PAGE gel. Lane 1, Coomassie blue–stained gel showing locations of histone bands. Lane 2, far western of a parallel lane using radiolabeled in vitro–translated probe. Lane 3, far western using unlabeled probe detected with probe-specific antibody. Lane 4, western blot using anti-histone H3 specific antibody. Lane 5, western blot using anti-histone H4 specific antibody.

View Image
Figure 17.2.2

Far western blot of peptides (A) dot blotted onto PVDF membrane and (B) stained with India ink. Figure reproduced with permission of Cold Spring Harbor Laboratory Press.

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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.