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Scintillation Proximity Assay (SPA) Technology to Study Biomolecular Interactions

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Abstract
Table of Contents
Materials
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Literature Cited

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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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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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⋅[3 H]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 [125 I]Bolton and Hunter reagent (unit 3.3 ), succinimidyl‐N‐[propionate‐2,3‐3 H] (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)
[125 I]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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Figure 19.8.1 Diagrammatic representation of SPA (not to scale). On the left, the bound radioligand is in close proximity, stimulating the bead to emit light. On the right, the unbound radioligand is not in sufficient proximity to stimulate light.

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Figure 19.8.2 Flow chart for the development of SPA protein‐protein interaction assays.

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Figure 19.8.3 The effect of varying the bead concentration on signal (B0 ) and NSB in the GRB2 assay. Assays were performed using 20 mM MOPS buffer, pH 7.5, containing 0.1% (w/v) BSA, 10 mM DTT, and varying concentrations of streptavidin coated SPA beads. The beads were settled and the plate was counted at 4°C. Results are means (n = 3).

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Figure 19.8.4 Diagrammatic representation of an SPA receptor binding assay (not to scale). Immobilized receptors on SPA beads are stimulated to emit light when a radioligand binds to the receptor. Any unbound radioligand is too distant from the bead to generate a signal.

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Figure 19.8.5 Flow chart for the development of SPA receptor binding assays.

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Figure 19.8.6 Competition binding curve showing the inhibition of [125 I]Tyr‐labeled PYY binding by unlabeled NPY(squares), PYY(upright triangles), [Leu31Pro34]NPY (upside‐down triangles), and NPY(13‐36) (diamonds), expressed as percent B/B0 . Results are means ± SEM (n = 3). Assay was performed using the conditions outlined (see ).

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Figure 19.8.7 Flow chart for the development of CAM interaction SPA.

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Figure 19.8.8 Time course for the E‐selectin SPA. Assay contained 0.1 mg/well SPA beads, 100 ng/well E‐selectin‐ZZ fusion protein, and 2 × 105 cpm sLex (20% molar ratio) [3 H] glycoconjugate. Assays were counted using a MicroBeta 1450 scintillation counter after incubation for 8 hr at 22°C. Data points are means ± range of duplicate wells.

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Figure 19.8.9 Titration of consensus DNA against NF‐κB‐GST‐p65. 25 nM GST‐p65, was titrated with up to 0.5 nM [3 H]DNA as described in the text. Results are means ± SD (n = 2) and are representative of three experiments.

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Figure 19.8.10 Flow chart for the development of SPA RIAs.

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Figure 19.8.11 The LEADseeker Components. (1 and 2) Light‐tight chambers containing CCD camera and microplate focusing mechanism. (3) Microplate stacker carousel. (4) Bar code reader. (5) Microplate shuttle mechanism. (6) Camera cooling unit.

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Figure 19.8.12 Effect of increasing GST‐NF1 and H‐RasL61⋅[3 H]GTP concentration on specific SPA cpm bound. 10 pmol/well H‐RasL61⋅[3 H]GTP (squares), 20 pmol/well H‐RasL61⋅[3 H]GTP (upward‐facing triangles), 30 pmol/well H‐RasL61⋅[3 H]GTP (downward‐facing triangles), 40 pmol/well H‐RasL61⋅[3 H]GTP (diamonds). Assay conditions: 3.75 µg anti‐GST antibody (Molecular Probes), 0.5 mg protein A beads in 50 mM Tris⋅Cl, pH 7.5, 2 mM DTT. Results are means ± SEM (n = 3).

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Figure 19.8.13 Inhibition of H‐RasL61⋅[3 H]GTP binding to GST‐NF1 by GTP (square), ATP (downward‐facing triangle), and GDP (upward‐facing triangle). Assay conditions as for Figure using 10 pmol H‐RasL61⋅[3 H]GTP and 40 pmol GST‐NF1. Results are means ± SEM (n = 2).

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Figure 19.8.14 Unlabeled peptides competing with peptide [3 H]108 for binding to the Crk SH3 domain. Assays were performed using 20 mM MOPS buffer, pH 7.5, containing 10 mM DTT, 1% (v/v) Triton X‐100, 0.1% (v/v) SDS, and 1 mg streptavidin SPA beads. Following overnight incubation at 2° to 8°C, the assays were counted using a TopCount (PerkinElmer). Results are means ± SEM (n = 3).

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Figure 19.8.15 Association/dissociation of [3 H]copolamine binding to mAChR1 measured by SPA. Stock solutions of mAChR1 membranes were rolled for 2 hr with WGA PVT SPA beads at a ratio of 50 µg membranes/4 mg beads. [3 H]Scopolamine (100 pM final) was added and the signal monitored by counting 1 min at 5‐min intervals using the MicroBeta 1450 scintillation counter (PerkinElmer). At equilibrium (after 2.5 hr), (−)‐quinuclidinyl benzilate (QNB) (2 µM final) was added and the decreasing signal measured as above. The data was fitted and kinetic constants estimated using EBDA (McPhearson, 1985).

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Literature Cited

Literature Cited
	Amersham Biosciences. 1997. Validation and characterisation of polyacrylamid‐based neoglycoconjugates as selectin ligands by scintillation proximity assay. Proximity News 3.
	Baeuerle, P.A. 1991. The inducible transcription activator NF‐κB: Regulation by distinct protein sub‐units. Biochim. Biophys. Acta 1072:63‐80.
	Beveridge, M., Park, Y.W., Hermes, J., Marenghi, A., Brophy, G., and Santos, A. 2000. Detection of p56(lck) kinase activity using scintillation proximity assay in 384‐well format and imaging proximity assay in 384‐ and 1536‐well format. J. Biomol. Screen. 5:205‐212.
	Baxendale, P.M., Martin, R.C., Hughes, K.T., Lee, D.Y., and Waters, N.M. 1990. Development of scintillation proximity assays for prostaglandins and related compounds. In Advances in Prostaglandins, Thromboxane and Leukotriene Research, Vol. 21 (B. Samuelsson et al., eds.) pp. 303‐306. Raven Press, New York.
	Bovin, N.V., Korchagina, E.Y., Zemlyanukhina, T.V., Byramova, N.E., Galanina, O.E., Zemlyakov, A.E., Ivanov, A.E., Zubov, V.P., and Mochalova, L.V. 1993. Synthesis of polymeric neoglycoconjugates based on N‐ substituted polyacrylamides. Glycocon. J. 10:142‐151.
	Buratowski, S

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