Detecting Low‐Affinity Extracellular Protein Interactions Using Protein Microarrays

Yi Sun1, Gavin J. Wright1

1 Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 27.5
DOI:  10.1002/0471140864.ps2705s72
Online Posting Date:  April, 2013
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Low‐affinity extracellular protein interactions are critical for cellular recognition processes, but are not generally detected by methods that can be applied in a high‐throughput manner. This unit describes a protein microarray platform that significantly improves the throughput of assays capable of detecting transient extracellular protein interactions. These methodological improvements now permit screening for novel extracellular receptor‐ligand interactions on a genome‐wide scale. Curr. Protoc. Protein Sci. 72:27.5.1‐27.5.15. © 2013 by John Wiley & Sons, Inc.

Keywords: receptor‐ligand pairs; extracellular protein interactions; AVEXIS; adhesion receptors; transient/weak interactions; high‐throughput screening; microarray

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Microarray AVEXIS Assay
  • Support Protocol 1: High‐Throughput Purification of 6His‐Tagged Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of Microarray AVEXIS Assay

  • Wash buffer (see recipe)
  • 20 µg each of the bait proteins at 400 ng/µl (see protocol 2)
  • Dilution buffer (see recipe)
  • Anti‐tag primary antibody [e.g., Anti‐Cd4 antibody (OX68); Santa Cruz, cat. no. sc‐53042]
  • Alkaline phosphatase‐conjugated secondary antibody
  • Substrate 104 (or other alkaline phosphatase substrate; Sigma‐Aldrich)
  • ELISA substrate buffer (see recipe)
  • 3 M NaOH
  • Microarray printing buffer (see recipe)
  • Nitrocefin (Calbiochen)
  • Anhydrous dimethyl sulfoxide (DMSO)
  • 1× Phosphate‐buffered saline (PBS; see recipe)
  • Milli‐Q water
  • Purified biotinylated HRP
  • Biotinylated anti‐prey antibody
  • Pin cleaning solution (Genomic Solutions; see recipe)
  • Microarray blocking buffer (see recipe)
  • HRP‐conjugated anti‐flag antibody
  • Tyramide signal amplification (TSA) kit (Life Technologies, cat. no. T30953) containing:
    • Labeled tyramide (Component A)
    • Hydrogen peroxide (Component F)
    • Amplification buffer (Component E)
  • N 2 gas, optional
  • Streptavidin‐coated 96‐well plate (Nunc)
  • Tissue paper
  • 22°C incubator
  • Aluminum foil
  • Plate reader (e.g., PHERAStar Plus; BMG Labtec)
  • 30,000‐MWCO (molecular weight cut off) spin concentrator
  • Streptavidin‐coated microarray slides (Xantec)
  • −20°C freezer
  • Centrifuge
  • V‐bottom 384‐well plates
  • Microarray spotting machine (MicroGrid II, Digilab)
  • FAST frame multi‐slide plate with slide holders (Whatman)
  • Vortex mixer

Support Protocol 1: High‐Throughput Purification of 6His‐Tagged Proteins

  • Tissue culture supernatants containing proteins of interest
  • 2 M imidazole, pH 7.4
  • 4 M NaCl
  • 20% ethanol
  • Deionized water
  • Running buffer (see recipe)
  • Elution buffer (see recipe)
  • 96‐well His MultiTrap HP Ni‐NTA plates (GE Healthcare)
  • Plate docking platform (Sun et al., )
  • 50‐ml plastic syringes
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Literature Cited

Literature Cited
   Bushell, K.M., Sollner, C., Schuster‐Boeckler, B., Bateman, A., and Wright, G.J. 2008. Large‐scale screening for novel low‐affinity extracellular protein interactions. Genome Res. 18:622‐630.
   Crosnier, C., Bustamante, L.Y., Bartholdson, S.J., Bei, A.K., Theron, M., Uchikawa, M., Mboup, S., Ndir, O., Kwiatkowski, D.P., Duraisingh, M.T., Rayner, J.C., and Wright, G.J. 2011. Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum. Nature 480:534‐537.
   Dustin, M.L., Golan, D.E., Zhu, D.M., Miller, J.M., Meier, W., Davies, E.A., and van der Merwe, P.A. 1997. Low affinity interaction of human or rat T cell adhesion molecule CD2 with its ligand aligns adhering membranes to achieve high physiological affinity. J. Biol. Chem. 272:30889‐30898.
   Kerr, J.S. and Wright, G.J. 2012. Avidity‐based extracellular interaction screening (AVEXIS) for the scalable detection of low‐affinity extracellular receptor‐ligand interactions. J. Vis. Exp. e3881.
   Letarte, M., Voulgaraki, D., Hatherley, D., Foster‐Cuevas, M., Saunders, N.J., and Barclay, A.N. 2005. Analysis of leukocyte membrane protein interactions using protein microarrays. BMC Biochem. 6:2.
   Martin, S., Sollner, C., Charoensawan, V., Adryan, B., Thisse, B., Thisse, C., Teichmann, S., and Wright, G.J. 2010. Construction of a large extracellular protein interaction network and its resolution by spatiotemporal expression profiling. Mol. Cell Proteomics 9:2654‐2665.
   Powell, G.T. and Wright, G.J. 2011. Jamb and Jamc are essential for vertebrate myocyte fusion. PLoS Biol. 9:e1001216.
   Ramani, S.R., Tom, I., Lewin‐Koh, N., Wranik, B., Depalatis, L., Zhang, J., Eaton, D., and Gonzalez, L.C. 2012. A secreted protein microarray platform for extracellular protein interaction discovery. Anal. Biochem. 420:127‐138.
   Sollner, C. and Wright, G.J. 2009. A cell surface interaction network of neural leucine‐rich repeat receptors. Genome Biol. 10:R99.
   Sun, Y., Gallagher‐Jones, M., Barker, C., and Wright, G.J. 2012. A benchmarked protein microarray‐based platform for the identification of novel low‐affinity extracellular protein interactions. Anal. Biochem. 424:45‐53.
   van der Merwe, P.A. and Barclay, A.N. 1994. Transient intercellular adhesion: the importance of weak protein‐protein interactions. Trends Biochem. Sci. 19:354‐358.
   Wojtowicz, W.M., Wu, W., Andre, I., Qian, B., Baker, D., and Zipursky, S.L. 2007. A vast repertoire of Dscam binding specificities arises from modular interactions of variable Ig domains. Cell 130:1134‐1145.
   Wright, G.J. 2009. Signal initiation in biological systems: The properties and detection of transient extracellular protein interactions. Mol. BioSystems 5:1405‐1412.
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