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SPA Assays for GTP Binding

Overview

In the SPA format, cell membranes are captured onto SPA scintillant beads. When the fGPCR is activated 35S-labeled GTP will bind to the membrane. This puts the radiochemical into proximity of the SPA bead. When the radiochemical is close to the bead, the beta energy from the 35S can interact with scintillant in the bead, producing a signal that can be measured. 35S-GTP that is not bound to the cell membrane will not be close enough to the SPA bead to interact strongly with the scintillant. 

spa-assays-for-gtp-binding-fig1

What do I need to run this assay?

  • Cell membrane expressing receptor of interest. (Revvity carrie receptor-transfected cell membranes)
  • 35S-gamma GTP (NEG030H or NEG030X)
  • Unlabeled non-hydrolyzable GTP-gamma-S control for non-specific binding
  • GDP, agonists, antagonists, test compounds as appropriate
  • WGA-coated SPA beads (See SPA bead options section for more information. We do not recommend using a PEI-coated bead (polyethyleneimine-coated bead) when working with 35S-gamma GTP.)
  • Microplates (We recommend Revvity white or white-walled, clear-bottom for bottom reads.)
  • TopSeal™-A (Cat. No. 6050185)
  • Appropriate detection instrument. (We recommend a MicroBeta™ or Tri-Carb™ liquid scintillation counter)

*Revvity also sells a GTP binding SPA kit, Cat. No. RPNQ0210. This kit contains WGA PVT SPA beads and assay buffer.

SPA bead options for capturing cell membranes

In addition to standard SPA scintillation beads (which can be measured on a Revvity MicroBeta Counter, we also offer SPA imaging beads. SPA imaging beads have a red-shifted signal output and can be measured on an appropriate instrument  

  • The most-commonly used SPA bead for this assay is the WGA-coated PVT scintillation SPA bead (#RPNQ0001)
  • In addition to the beads below, we also carry kit #RPNQ0210 which contains WGA-coated PVT beads and buffer. This kit contains 750 mg of WGA PVT SPA beads and a 5X Assay Buffer concentrate (100 mM HEPES, 500 mM NaCl, 50 mM MgCl2, 5 mM EDTA, pH 7.4 prior to dilution).
Bead coating Bead type Core bead type Pack size Catalog number
Wheat germ agglutinin (WGA)  Scintillation       PVT 100mg RPNQ0252
  500mg RPNQ0001
  25 x 500 mg SPQ0031
  2g RPNQ0060
  YSI  250 mg RPNQ0011
  1 g RPNQ0023
  PS 50 mg RPNQ0262
  500 mg RPNQ0260
  VOX 50 mg RPNQ0272
  500 mg RPNQ0270
     
spa-assays-for-gtp-binding-fig2

35S-gamma GTP radiochemicals

Two different 35S-gamma GTP products are available from Revvity, each in various sizes.

Product number Radioactive concentration Specific activity Buffer
NEG030H 12.5 mCi/mL 1250 Ci/mmol 10 mM Tricine pH 7.6, 10 mM DTT
NEG030X 1 mC/mL 1250 Ci/mmol 10 mM Tricine pH 7.6, 10 mM DTT

 

Protocol-in-brief

This diagram shows how a SPA GTP binding assay works in general  

spa-assays-for-gtp-binding-fig3

Assay optimization

1. Concentration of GDP, MgCl in assay (cross-titration matrix):

Test 4 different concentrations:

  • For MgCl2: 1 mM, 3 mM, 10 mM, and 30 mM
  • For GDP: 1 µM, 3 µM, 10 µM and 30 µM


Work in triplicate:

  • 3 basal wells (no agonist)
  • 3 stimulated wells (with a high concentration of agonist)


As a general recommendation, you can run this experiment using 10 μg of membrane and 0.5 mg of beads per well (96-well format, 100 µL reaction volume).

2. Cell membrane and SPA bead titration (cross-titration matrix):

  • Try three random membrane concentrations [for example: 5, 10 and 15 µg per well (100 µL reaction volume)] and three concentrations of beads [for example: 1, 0.5 and 0.25 mg beads per well (100 µL reaction volume)]. Set up both basal and stimulated samples.

3. Agonist dose-response under optimized conditions (antagonist dose response, Z' values if applicable).

  • Perform a 10x serial dilution of your agonist (you want to cover a broad range of agonist concentrations). For antagonist dose-response, use agonist at concentration equivalent to the EC80. 

Recommended assay conditions (SPA format) for 35S-gamma-GTP binding assays using Revvity cell membrane products validated for GTP binding

Cat #   Assay buffer Membranes 
(µg final)		 GDP 
(µM final)		 PVT-WGA SPA Beads 
(mg final)		 GTPg35S 

(dpm)
ES-010-M400UA Adenosine, A1 20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2 2.5 µg 10 µM 0.5 mg 25.000 dpm
ES-012-M400UA Adenosine, A3 20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2  4 µg 1 µM 0.25 mg 25.000 dpm
ES-030-M400UA Adrenergic, alpha 2A 20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2 5 µg 10 µM 0.25 mg 25.000 dpm
ES-031-M400UA Adrenergic, alpha 2B 20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2 10 µg 15 µM 0.5 mg 25000 dpm
ES-110-M400UA Cannabinoid, CB1 20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2, 0.1% protease free BSA 5 µg 3 µM 0.5 mg 25.000 dpm
ES-111-M400UA Cannabinoid, CB2 20 mM HEPES pH 7.4, 200 mM NaCl, MgCl2, 0.1% protease free BSA. 5 µg 3 µM 0.5 mg 25.000 dpm
ES-137-M400UA Chemokine, CX3CR1 20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2 5 µg 1 µM 0.25 mg 25.000 dpm
ES-142-M400UA Chemokine, CXCR3 20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2 5 µg 3 µM 0.25 mg 25.000 dpm
ES-145-M400UA Chemokine, CXCR2 20 mM HEPES pH 7.4, 100 mM NaCl, 3 mM MgCl2, 0.1% protease-free BSA. 10 µg 3 µM 0.5 mg 25.000 dpm
ES-148-M400UA Chemokine, CXCR1 20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2, 0.1% protease free BSA. 10 µg 3 µM 0.5 mg 25.000 dpm
ES-173-M400UA Dopamine, D3 20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2, 0.1% protease free BSA. 10 µg 1 µM 0.5 mg 25.000 dpm
ES-340-M400UA Leukotriene, BLT1 20 mM HEPES pH 7.4; 100 mM NaCl, 30 mM MgCl2 10 µg 10 µM 0.5 mg 25.000 dpm
ES-370-M400UA MCH 1/ SCL1 20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2, 0.1% protease free BSA. 4 µg 10 µM 0.5 mg 25.000 dpm
ES-392-M400UA Histamine, H3 20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2 5 µg 10 µM 0.5 mg 25.000 dpm
ES-491-M400UA Neuropeptide, NPFF1 20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2, 0.1% protease free BSA. 10 µg 10 µM 0.25 mg 25.000 dpm
ES-511-M400UA Galanin, Gal 2 20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2 5 µg 1 µM 5 mg 25.000 dpm
ES-530-M400UA Cholecystokinin, CCK1 20 mM HEPES pH 7.4, 100 mM NaCl, 3 mM MgCl2, 0.1% protease free BSA 5 µg 1 µM 0.5 mg 25.000 dpm
ES-521-M400UA Somatostatin, sst2a 20 mM HEPES pH 7.4, 100 mM NaCl, 3 mM MgCl2. 5 µg 3 µM 0.5 mg 25.000 dpm
ES-522-M400UA Somatostatin, sst5 20 mM HEPES pH 7.4, 100 mM NaCl, 3 mM MgCl2 8 µg 3 µM 0.25 mg 25.000 dpm
ES-523-M400UA Somatostatin, sst3 20 mM HEPES pH 7.4, 100 mM NaCl, 3 mM MgCl2 10 µg 3µM 0.25 mg 25.000 dpm
ES-541-M400UA Opioid, Kappa (OP2) 20 mM HEPES pH 7.4, 100 mM NaCl, 30 mM MgCl2, 0.1% protease free BSA. 5 µg 10 µM 0.25 mg 25.000 dpm
ES-542-M400UA Opioid, Mu (OP3) 20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2, 5 µg 1 µM 0.5 mg 25.000 dpm
ES-561-M400UA Prostanoid, CRTH2 20 mM HEPES pH 7.4; 100 mM NaCl, 30 mM MgCl2, 0.1% protease free BSA. 10 µg 10 µM 0.5 mg 25.000 dpm
ES-610-M400UA N-formyl Peptide, FPRL1 20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2, 0.1% protease free BSA. 10 µg 30 µM 0.25 mg 25.000 dpm
ES-620-M400UA Melatonin, MT1 20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2 10 µg 10 µM 0.25 mg 25.000 dpm
ES-621-M400UA Melatonin, MT2 20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2 10 µg 50 µM 0.25 mg 25.000 dpm
ES-720-M400UA Chemokine, CXCR6 20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2 10 µg 3 µM 0.5 mg 25000 dpm
ES-730-M400UA Chemotactic Peptide, C3a 20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2, 0.1% protease free BSA. 5 µg 30 µM 0.25 mg 25.000 dpm

 

Application note, posters, guides and other resources

Tips and FAQs

  • Cell membrane concentration and bead concentration are critical in SPA assays - these parameters need to be optimized carefully for each assay
  • For GTP binding assays, we do not recommend using PEI-coated (polyethyleneimine-coated) SPA beads.
  • The GTPγS assay works best with Gi-coupled GPCRs. Very low assay windows are usually obtained for Gs- and Gq-coupled receptors, due to both (1) levels of expression of Gi relative to Gs or Gq proteins and (2) the exchange rate of these G proteins for GTP.
  • The GTPγS assay is sensitive to GDP concentration, concentration of 35S GTPγS, Mg2+ in the assay buffer.
  • Controls: we recommend that when you develop your assay, you include a no-agonist control (substituting with buffer) to determine your basal GTP binding level, and a non-specific binding (NSB) control where you use unlabeled non-hydrolyzable GTP with 35S-gamma-GTP and cell membrane

Data analysis

Visit our GTP binding data analysis page.

Citations

  1. Wan, Y. et al. Identification of full, partial, and inverse CC chemokine receptor 3 agonists using 35S GTPgammaS binding. Eur. J. Pharmacol 456, 1-10 (2002). Link
  2. Rodgers, G. et al. Development of displacement binding and GTPgammaS scintillation proximity assays for the identification of antagonists of the micro-opioid receptor. Assay Drug Dev Technol 1, 627-636 (2003). Link
  3. Johnson, E.N. et al. A 1,536-well 35S GTPgammaS scintillation proximity binding assay for ultra-high-throughput screening of an orphan galphai-coupled GPCR. Assay Drug Dev Technol 6, 327-337 (2008). Link
  4. Ferrer, M. et al. A fully automated 35S GTPgammaS scintillation proximity assay for the high-throughput screening of Gi-linked G protein-coupled receptors. Assay Drug Dev Technol 1, 261-273 (2003). Link

Custom cell lines, membranes, frozen cells, and receptors

Revvity offers custom radiosynthesis, labeling, cell lines and membranes as well as custom assay development. If you are interested in our custom services, please contact us.

Radiosynthesis and Labeling Custom Assay Services

For research use only. Not for use in diagnostic procedures. 

The information provided above is solely for informational and research purposes only. Revvity assumes no liability or responsibility for any injuries, losses, or damages resulting from the use or misuse of the provided information, and Revvity assumes no liability for any outcomes resulting from the use or misuse of any recommendations. The information is provided on an "as is" basis without warranties of any kind. Users are responsible for determining the suitability of any recommendations for the user’s particular research. Any recommendations provided by Revvity should not be considered a substitute for a user’s own professional judgment.