Scintillation Proximity Assay

Steven D. Kahl1, Christian C. Felder1

1 Lilly Research Laboratories, Indianapolis, Indiana
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.15
DOI:  10.1002/0471142301.ns0715s30
Online Posting Date:  February, 2005
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Abstract

Scintillation proximity assay technologies provide a rapid non‐separation method to measure common biological interactions using radioactively tagged molecules. This unit identifies potential uses of the technology for the measurement of receptor‐ligand binding, cAMP accumulation, GTP binding to heterotrimeric G proteins, protease activity and cellular uptake.

     
 
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Table of Contents

  • Basic Protocol 1: Saturation Analysis of [3H]5‐HT Binding to the 5‐HT1E Receptor
  • Support Protocol 1: Optimization of Membrane Protein Concentration
  • Support Protocol 2: Optimization of Amount of WGA SPA Beads
  • Support Protocol 3: Optimization of Incubation Time
  • Basic Protocol 2: Pharmacological Profile for the 5‐HT1E Receptor Using SPA
  • Basic Protocol 3: Rapid Measurement of cAMP Accumulation in CHO Cells Stably Expressing the Muscarinic M1 Receptor
  • Basic Protocol 4: Time‐Course Analysis of Rhinovirus 3C Protease Using SPA
  • Basic Protocol 5: Uptake of [14C]p‐Hydroxy‐Loracarbef Using Cytostar‐T Plates
  • Basic Protocol 6: Muscarinic M1 Receptor–Mediated [γ‐35S]GTP Binding: An SPA Approach Using a Specific Anti–G Protein Antibody
  • Basic Protocol 7: Miniaturization of Receptor‐Radioligand Interactions Using 384‐Well FlashPlates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Saturation Analysis of [3H]5‐HT Binding to the 5‐HT1E Receptor

  Materials
  • LM(tk‐) cells expressing 5‐HT 1E receptor (Kahl et al., )
  • 50 mM Tris⋅Cl, pH 7.4 ( appendix 2A), ice‐cold
  • Bovine serum albumin (BSA)
  • Saturation assay buffer (see recipe), room temperature
  • 20 mg/ml WGA SPA beads (see recipe)
  • 1 mM 5‐hydroxytryptamine (unlabeled 5‐HT; see recipe)
  • 1 µM [3H]5‐hydroxytryptamine ([3H]5‐HT; see recipe)
  • Teflon pestle/glass tissue homogenizer (e.g., Thomas)
  • 250‐ml conical centrifuge tubes (e.g., Corning)
  • Beckman refrigerated tabletop centrifuge, or equivalent
  • Beckman J2‐21M centrifuge and JA‐14 rotor with 250‐ml bottles, or equivalent
  • 13 × 100–mm disposable polypropylene tubes
  • 96‐well white polystyrene microtiter plates (e.g., clear‐bottom from Corning or opaque bottom from Perkin Elmer Life and Analytical Sciences)
  • Single and multichannel pipettors
  • Microtiter plate shaker (e.g., Labline Instruments)
  • Self‐adhesive microtiter plate sealers
  • Liquid scintillation cocktail (e.g., Ready Protein+, Beckman)
  • Liquid scintillation counter (e.g., Packard Tricarb) and glass scintillation vials
  • Microtiter plate scintillation counter (e.g., Perkin Elmer Life and Analytical Sciences Trilux or TopCount)
  • Additional reagents and equipment for protein assays (see CPMB UNIT ) and nonlinear regression analysis (unit 7.5)

Support Protocol 1: Optimization of Membrane Protein Concentration

  Materials
  • Serotonergic compounds (also see Table 7.15.4), e.g.:
    • 10 mM α‐methyl‐5‐hydroxytryptamine (see recipe)
    • 10 mM 2‐methyl‐5‐hydroxytryptamine (see recipe)
    • 10 mM 5‐carboxyamidotryptamine (see recipe)
  • 1 ml volume/well 96‐well polypropylene microtiter plate
  • 50‐ml conical disposable polypropylene tubes
  • Additional reagents and equipment for saturation analysis of [3H]5‐HT binding to the 5‐HT 1E receptor (see protocol 1)

Support Protocol 2: Optimization of Amount of WGA SPA Beads

  Materials
  • CHO cells (ATCC CCL‐61) stably expressing the human M 1 muscarinic receptor (Felder et al., )
  • DMEM/ F‐12/10% FBS medium (see recipe) with HEPES
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Trypsin/EDTA solution: 0.05% trypsin/0.53 mM tetrasodium EDTA (e.g., Life Technologies)
  • Enzyme (trypsin)‐free cell dissociation solution (Specialty Media)
  • Dilution buffer (see recipe)
  • 1 mM 3‐isobutyl‐1‐methylxanthine (IBMX; Sigma) or 10 µM RO20‐1724 (Calbiochem) in dilution buffer
  • Carbachol (Research Biochemicals)
  • Lysis buffer: 1% (w/v) dodecyltrimethylammonium bromide in 50 mM sodium acetate, pH 5.8
  • 500 µM adenosine 3′:5′‐cyclic monophosphate (cAMP), sodium salt (Sigma)
  • 50 mM sodium acetate buffer, pH 5.8
  • Beads/antibody/[125I]cAMP mixture (see recipe)
  • 75‐cm2 and 225‐cm2 tissue culture flasks (Costar)
  • 96‐well microtiter plates (white plate; clear bottom; Fisher or Costar)
  • Microtiter plate sealing tape (Wallac)
  • Microtiter plate scintillation counter (e.g., Wallac Trilux or Packard TopCount)
  • Additional reagents and equipment for cell culture and counting cells ( appendix 3B)
NOTE: All culture incubations are performed in a 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Support Protocol 3: Optimization of Incubation Time

  Materials
  • Protease assay buffer (see recipe)
  • Stop solution: 10% (w/v) orthophosphoric acid
  • 50 nM biotinylated 125I‐labeled rhinovirus 3C protease substrate stock solution (see recipe)
  • 50 µM purified rhinovirus 3C protease stock (store at −80°C; Birch et al., )
  • 20 mg/ml streptavidin SPA beads (see recipe), 4°C
  • 13 × 100–mm disposable polypropylene tubes
  • 96‐well white polystyrene microtiter plates (e.g., clear bottom from Corning or opaque bottom from Perkin Elmer Life and Analytical Sciences)
  • Single and multichannel pipettors
  • Microtiter plate shaker (e.g., Labline Instruments)
  • Self‐adhesive microplate sealers
  • Microtiter plate scintillation counter (e.g., Perkin Elmer Life and Analytical Sciences Trilux or TopCount)

Basic Protocol 2: Pharmacological Profile for the 5‐HT1E Receptor Using SPA

  Materials
  • Human adenocarcinoma cell line Caco‐2 (obtained from Dr. J. Fogh at the Research Unit of Memorial Sloan‐Kettering Cancer Center, Rye, NY)
  • DMEM/F12/10% FBS medium without HEPES (see recipe)
  • Trypsin/EDTA solution: 0.05% trypsin/0.53 mM tetrasodium EDTA (e.g., Life Technologies; store at 4°C)
  • 4 mg/ml rat tail collagen, type II (see recipe)
  • 0.5% (v/v) acetic acid
  • Ammonium hydroxide
  • 20 mM [14C]p‐hydroxy‐loracarbef (see recipe)
  • Flux buffer (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 75‐cm2 sterile tissue culture flasks (sterile, tissue culture treated; Costar)
  • Cytostar‐T microtiter plate (sterile, tissue culture treated; Amersham Biosciences)
  • Multichannel pipettor
  • 8‐channel aspirator
  • Self‐adhesive microplate seals
  • Microtiter plate scintillation counter (e.g., Perkin Elmer Life and Analytical Sciences Trilux or TopCount)
  • Additional reagents and equipment for cell culture and counting cells ( appendix 3B)
NOTE: All culture incubations are performed in a 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 3: Rapid Measurement of cAMP Accumulation in CHO Cells Stably Expressing the Muscarinic M1 Receptor

  Materials
  • Membrane preparation from CHO cells expressing recombinant human M 1 muscarinic receptor (Perkin‐Elmer Life and Analytical Sciences)
  • Oxotremorine‐M (Sigma)
  • Dimethyl sulfoxide (DMSO)
  • Compound dilution buffer: muscarinic assay buffer (see recipe) containing 4% (v/v) DMSO (add 2 ml DMSO to 48 ml assay buffer; prepare fresh each day)
  • Muscarinic assay buffer (see recipe)
  • [γ‐35S]GTP in stable aqueous solution (Perkin‐Elmer Life and Analytical Sciences), store up to 60 days at −20°C (see recipe for preparing [γ‐35S]GTP ligand solution)
  • 10% (v/v) Nonidet P‐40 (NP‐40; Roche), 4°C
  • Rabbit anti‐Gα q/11 antibody (Santa Cruz Biotechnology; see recipe for preparing diluted antibody)
  • SPA PVT antibody‐binding beads, anti‐rabbit reagent (Amersham Biosciences; store according to manufacturer's recommendations)
  • 50‐ml disposable polystyrene tubes
  • 10‐ml disposable glass tubes
  • 96‐well deep‐well polypropylene microtiter plate (1 ml/well capacity)
  • Tissue homogenizer (e.g., PowerGen 700, Fisher)
  • 50‐ml polystyrene reagent reservoirs
  • Single and multichannel pipettors
  • 96‐well polystyrene microtiter plate (white, with clear bottom; Corning Costar)
  • Self‐adhesive microtiter plate seals
  • Microtiter plate shaker (e.g., Vortex Genie, Scientific Industries)
  • Tabletop centrifuge (e.g., AccuSpin 1R, Fisher) with microtiter plate carrier
  • Microtiter plate scintillation counter (e.g., Perkin‐Elmer Life and Analytical Sciences Trilux)

Basic Protocol 4: Time‐Course Analysis of Rhinovirus 3C Protease Using SPA

  Materials
  • Miniature SPA assay buffer (see recipe)
  • 1.5 mM S(–)‐propranolol (see recipe)
  • 500 nM [125I]iodocyanopindolol (see recipe)
  • Membrane preparation (2.0 µg/ml) from HEK293 cells expressing β 2 adrenergic receptor (Perkin Elmer Life and Analytical Sciences)
  • 13 × 100–mm disposable polypropylene tubes
  • FlashPlates (Perkin Elmer Life and Analytical Sciences, store at 4°C):
    • 96‐well Basic (uncoated) FlashPlate
    • 96‐well WGA FlashPlate
    • 384‐well Basic (uncoated) FlashPlate
    • 384‐well WGA FlashPlate
  • Multichannel pipettors and corresponding reagents reservoirs
  • Gamma counter (e.g., ICN Micromedic)
  • Microtiter plate shaker (e.g., Vortex Genie, Scientific Instruments)
  • Self‐adhesive microtiter plate seals
  • Microplate scintillation counter (e.g., Perkin Elmer Life and Analytical Sciences Trilux or TopCount)
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Figures

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