Identification of Protein Interactions by Far Western Analysis

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

This unit describes far western blotting, a method of identifying protein?protein interactions. In a far western blot, one protein of interest is immobilized on a solid support membrane, then probed with a non?antibody protein. As described, far western blots can be used to identify specific interacting proteins in a complex mixture of proteins. They are particularly useful for examining interactions between proteins that are difficult to analyze by other methods due to solubility problems or because they are difficult to express in cells. This method is performed totally in vitro, and the proteins of interest can be prepared in a variety of ways. A protocol is also provided for determining the effects of specific peptide residues or post?translational modifications on protein?protein interactions. Many different detection techniques, either radioactive or nonradioactive, can be used. For example, the protein probe may be detected indirectly with an antibody, rather than being labeled radioactively.

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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 (unit 10.1 )
Ponceau S staining solution (see recipe )
Blocking buffer I: 0.05% (w/v) Tween 20 in recipe1× PBS (see recipe for recipePBS ); prepare fresh
Blocking buffer II: dissolve 1 g bovine serum albumin (BSA; fraction V) in 100 ml recipe1× PBS (see recipe for recipePBS ); prepare fresh
recipePhosphate‐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 ³⁵ S‐methionine (1000 Ci/mmol)
recipeProbe purification buffer (see recipe )
recipeProbe dilution buffer (see 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 10.1 ), electrophoretic transfer of proteins to a support membrane (unit 10.7 ), and autoradiography (unit 10.11 )

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 recipe1× TBST (see recipe for recipeTBST )
Primary antibody specific for protein probe
recipeTBST (see recipe )
Alkaline phosphatase (AP)–conjugated secondary antibody against Ig of species from which specific antibody was obtained
recipeAlkaline phosphatase buffer (see recipe )
recipeDeveloping solution (see recipe )
100 mM EDTA, pH 8.0 ( appendix 2E )

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

Peptides
recipe0.4% Tween 20/PBS (see recipe )
recipeIndia 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 19.7.1 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.

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Figure 19.7.2 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.
	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.

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Identification of Protein Interactions by Far Western Analysis

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited