Scintillation Proximity Assay (SPA) Technology to Study Biomolecular Interactions

Neil Cook1, Alison Harris1, Alison Hopkins1, Kelvin Hughes1

1 Amersham Biosciences Ltd., Cardiff
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.8
DOI:  10.1002/0471140864.ps1908s27
Online Posting Date:  May, 2002
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Abstract

Scintillation proximity assay (SPA) is a versatile homogeneous technique for radioactive assays which eliminates the need for separation steps. In SPA, scintillant is incorporated into small fluomicrospheres. These microspheres or “beads” are constructed in such a way as to bind specific molecules. If a radioactive molecule is bound to the bead, it is brought into close enough proximity that it can stimulate the scintillant contained within to emit light. Otherwise, the unbound radioactivity is too distant, the energy released is dissipated before reaching the bead, and these disintegrations are not detected. In this unit, the application of SPA technology to measuring protein‐protein interactions, Src Homology 2 (SH2) and 3 (SH3) domain binding to specific peptide sequences, and receptor‐ligand interactions are described. Three other protocols discuss the application of SPA technology to cell‐adhesion‐molecule interactions, protein‐DNA interactions, and radioimmunoassays. In addition, protocols are given for preparation of SK‐N‐MC cells and cell membranes.

     
 
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Table of Contents

  • Basic Protocol 1: The Application of SPA Technology to the Measurement of Protein‐Protein Interactions
  • Basic Protocol 2: The Application of SPA Technology to Src Homology 2 (SH2) and Src Homology 3 (SH3) Domain Binding to Specific Peptide Sequences
  • Basic Protocol 3: The Application of SPA Technology to Study Receptor‐Ligand Interactions
  • Support Protocol 1: SK‐N‐MC Cell Culture and Cell Harvesting
  • Support Protocol 2: Preparation of Cell Membranes
  • Basic Protocol 4: The Application of SPA Technology to Cell Adhesion Molecule (CAM) Interaction Assay
  • Basic Protocol 5: The Application of SPA Technology to Study Protein/DNA Interactions
  • Basic Protocol 6: The Application of SPA Technology to Radioimmunoassays (RIAs)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Application of SPA Technology to the Measurement of Protein‐Protein Interactions

  Materials
  • GST‐NF1 protein (Eccleston et al., )
  • recipeNF1‐Ras SPA buffer (see recipe)
  • RasL61⋅[3H]GTP (Lowe et al., ; also see )
  • Anti‐GST antibody (Molecular Probes)
  • 500 mg/vial protein A PVT SPA beads, lyophilized (Amersham Biosciences; Table 19.8.1)
  • Test compound and appropriate solvent
  • Siliconized glass vial ( appendix 3E)
  • 96‐well microtiter plate and adhesive sealer or assay tubes and caps
  • 22°C water bath or microtiter plate incubator

Basic Protocol 2: The Application of SPA Technology to Src Homology 2 (SH2) and Src Homology 3 (SH3) Domain Binding to Specific Peptide Sequences

  Materials
  • recipeGrb2‐SH2/SH3 SPA buffer (see recipe)
  • recipeBiotin/streptavidin capture buffer (see recipe)
  • 500 mg/vial freeze‐dried protein A or streptavidin PVT SPA beads (Amersham Biosciences)
  • Test compound and appropriate solvent (e.g., DMSO; optional)
  • GST‐SH2/SH3 fusion protein: express in E. coli, purify by chromatography on glutathione‐Sepharose 4B, and filter centrifuge using a Centriprep 10 microconcentration device (Amicon)
  • Anti‐GST antibody (Molecular Probes)
  • 96‐well microtiter plates with adhesive sealer or assay tubes with caps
  • Additional reagents and equipment for synthesizing peptides by solid‐phase Fmoc chemistry (unit 18.5), radiolabeling peptides using either [125I]Bolton and Hunter reagent (unit 3.3), succinimidyl‐N‐[propionate‐2,3‐3H] (Tang et al., ), or catalytic reduction under tritium gas (Evans, 1966), and purifying peptides by reversed‐phase HPLC (unit 11.6)
NOTE: This example is an optimized protocol containing amounts of the various assay components that give an appropriate counting window. In order to optimize each component, assays should be performed by varying the final concentration of the component under investigation, while keeping the concentration of the others fixed. For example, Figure shows the effect of varying the amount of the SPA beads in a Grb2 SH2 assay.

Basic Protocol 3: The Application of SPA Technology to Study Receptor‐Ligand Interactions

  Materials
  • recipeYY SPA buffer, 2° to 8°C (see recipe)
  • 500 mg/vial lyophilized WGA PVT SPA beads (Amersham Biosciences)
  • [125I]Tyr‐labeled peptide YY (Amersham Biosciences)
  • 500 µg/ml unlabeled porcine peptide YY (Bachem): prepare as recommended by the manufacturer; store up to at least two weeks at −15° to −30°C
  • Test compound and appropriate solvent
  • SK‐N‐MC cell (see protocol 4) or membrane preparation (see protocol 5)
  • 96‐well microtiter plate with adhesive seal or assay tubes with caps
NOTE: SPA is an homogeneous technique, and as such it is important that the assay be allowed to reach equilibrium before counting is performed. This normally requires a more prolonged incubation period than traditional filtration techniques. As the assay incubation is longer, it is therefore essential to ensure that the assay components are themselves stable. This may involve the addition of stabilizing agents or protease inhibitors to protect the ligand and/or receptor from the effect of degrading enzymes present in the receptor preparation. In addition, certain other cofactors such as divalent cations may be required for the binding of the radioligand to its receptor.

Support Protocol 1: SK‐N‐MC Cell Culture and Cell Harvesting

  Materials
  • SK‐N‐MC human neuroblastoma cells (ATCC# HTB‐10): store as recommended by ATCC
  • Complete Dulbecco's modified Eagle medium containing 10% (v/v) FBS (DMEM‐10; e.g., Sigma; also see appendix 3C)
  • Dulbecco's PBS, sterile (Life Technologies)
  • Trypsin/EDTA (Life Technologies)
  • recipeHarvesting buffer, cold (see recipe)
  • Cell scraper
  • 168‐cm2 flasks
  • 50‐ml centrifuge tube
NOTE: SK‐N‐MC cells do not grow well when cultured in roller bottles. The use of large tissue culture flasks is therefore recommended so that the cells remain permanently immersed in culture medium.

Support Protocol 2: Preparation of Cell Membranes

  Materials
  • Confluent monolayers of Chinese hamster ovary (CHO) cells genetically modified to express M 1 muscarinic acetylcholine receptors (mAChR1; ATTC# CRL‐1985)
  • Dulbecco's PBS, cold (Life Technologies)
  • recipeHypotonic buffer, ice‐cold (see recipe)
  • recipeDilution buffer (see recipe)
  • Trypsin/EDTA (Life Technologies)
  • Cell scraper
  • 50‐ml centrifuge tube
  • Hand‐held Dounce homogenizer and tight‐fitting pestle with 0.07 mm clearance (Bellco Glass)
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Figures

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Literature Cited

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