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SPA Assays for GTP Binding

Overview


In the SPA format, cell membranes are captured onto SPA scintillant beads. When the GPCR 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.

spagtp2_ASK.jpg
SPA GTP binding assay

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What do I need to run this assay?


  • Cell membrane expressing receptor of interest. (PerkinElmer carries 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 PerkinElmer white Optiplates™ or white-walled, clear-bottom IsoPlates™ for bottom reads.)
  • TopSeal-A™ (Cat. No. 6050195)
  • Appropriate detection instrument. (We recommend a TopCount® counter or MicroBeta® counter or Tri-Carb® liquid scintillation counter for SPA scintillation beads, or a ViewLux™ CCD Imager for SPA imaging beads.)

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

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SPA bead options for capturing cell membranes


In addition to standard SPA scintillation beads (which can be measured on a PerkinElmer TopCount or MicroBeta Counter, we also offer SPA imaging beads. SPA imaging beads have a red-shifted signal output, and can be measured on an instrument such as a PerkinElmer ViewLux Imager or a LEADseeker (GE).

  • 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 coatingBead typeCore bead typePack sizeCatalog number
Wheat germ agglutinin (WGA)      Scintillation      PVT    100 mgRPNQ0252
500 mgRPNQ0001
25 x 500 mgSPQ0031
2 gRPNQ0060
25 x 2 gRPNQ0063
Ysi 250 mgRPNQ0011
1 gRPNQ0023
Wheat germ agglutinin (WGA)      Imaging      PS   50 mgRPNQ0262
500 mgRPNQ0260
25 x 500 mgRPNQ0281
2 gRPNQ0308
Yox  50 mgRPNQ0272
500 mgRPNQ0270
25 x 500 mgRPNQ0282

spabeadtypes1_ASK.jpg
SPA bead types

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35S-gamma GTP radiochemicals


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

Product numberRadioactive concentrationSpecific activityBuffer
NEG030H12.5 mCi/mL1250 Ci/mmol10 mM Tricine pH 7.6, 10 mM DTT
NEG030X1 mC/mL1250 Ci/mmol10 mM Tricine pH 7.6, 10 mM DTT

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Protocol-in-brief


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


A detailed sample protocol is provided for a SPA GTP binding assay using cell membranes from our adenosine A1 receptor-transfected cells (#ES-010-C). Please keep in mind that the optimal concentrations of GDP, MgCl2, saponin, beads, and cell membrane will vary from receptor to receptor.

spa_gtp_workflow_GTP.jpg
Protocol-in-brief

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Assay optimizations


1. Concentration of GDP, MgCl2 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.

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Recommended assay conditions (SPA format) for 35S-gamma-GTP binding assays using PerkinElmer 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-M

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-M

20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2

4 µg

1 µM

0.25 mg

25.000 dpm

ES-030-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

5 µg

10 µM

0.25 mg

25.000 dpm

ES-031-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

10 µg

15 µM

0.5 mg

25000 dpm

ES-110-M

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-M

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-132-M

20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2

5 µg

3 µM

0.5 mg

25.000 dpm

ES-134-M

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-135-M

20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2

10 µg

3 µM

0.5 mg

25.000 dpm

ES-136-M

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-137-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

5 µg

1 µM

0.25 mg

25.000 dpm

ES-138-M

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-139-M

20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2, 0.1% protease free BSA.

10 µg

3 µM

0.5 mg

26.800 dpm

ES-140-M

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-142-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

5 µg

3 µM

0.25 mg

25.000 dpm

ES-145-M

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-M

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-M

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-210-M

20 mM HEPES pH 7.4; 100 mM NaCl, 30 mM MgCl2

10 µg

3 µM

0.25 mg

25.000 dpm

ES-211-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

2.5 µg

3µM

0.5 mg

25.000 dpm

ES-213-M

20 mM HEPES pH 7.4; 100 mM NaCl, 10 mM MgCl2, 0.1% protease free BSA.

4 µg

3 µM

0.25 mg

25.000 dpm

ES-230-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

5 µg

3 µM

0.5 mg

25.000 dpm

ES-310-M

20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2

5 µg

3 µM

0.5 mg

25.000 dpm

ES-340-M

20 mM HEPES pH 7.4; 100 mM NaCl, 30 mM MgCl2

10 µg

10 µM

0.5 mg

25.000 dpm

ES-370-M

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-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

5 µg

10 µM

0.5 mg

25.000 dpm

ES-393-M

20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2, 0.1% protease free BSA.

10 µg

10 µM

0.5 mg

25000 dpm

ES-460-M

(20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

5 µg

3 µM

0.5 mg

25.000 dpm

ES-490-M

20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2

5 µg

3 µM

0.25 mg

25.000 dpm

ES-491-M

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-500-M

20 mM HEPES pH 7.4; 100 mM NaCl, 3 mM MgCl2, 0.1% protease free BSA.

10 µg

3 µM

0.25 mg

25000 dpm

ES-511-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

5 µg

1 µM

5 mg

25.000 dpm

ES-520-M

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-M

20 mM HEPES pH 7.4, 100 mM NaCl, 3 mM MgCl2

5 µg

3 µM

0.25 mg

25.000 dpm

ES-522-M

20 mM HEPES pH 7.4, 100 mM NaCl, 3 mM MgCl2

8 µg

3 µM

0.25 mg

25.000 dpm

ES-523-M

20 mM HEPES pH 7.4, 100 mM NaCl, 3 mM MgCl2

10 µg

3 µM

0.25 mg

25.000 dpm

ES-524-M

20 mM HEPES pH 7.4; 100 mM NaCl, 10 mM MgCl2, 0.1% protease free BSA.

5 µg

10 µM

0.25 mg

25.000 dpm

ES-541-M

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-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2,

5 µg

1 µM

0.5 mg

25.000 dpm

ES-561-M

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-M

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-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

10 µg

10 µM

0.25 mg

25.000 dpm

ES-621-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

10 µg

50 µM

.25 mg

25.000 dpm

ES-656-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

10 µg

100 µM

0.5 mg

25.000 dpm

ES-720-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

10 µg

3 µM

0.5 mg

25.000 dpm

ES-730-M

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

ES-731-M

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

ES-760-M

20 mM HEPES pH 7.4; 100 mM NaCl, 1 mM MgCl2

7.5 µg

30 µM

0.25 mg

25.000 dpm

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Application note, posters, guides and other resources


  • Poster comparing filtration and scintillation bead SPA GTP binding assays using rat adenosine A1 receptor membranes
  • Sample protocol for a SPA GTP binding assay using cell membranes from our adenosine A1 receptor-transfected cells (#ES-010-C)
  • NIH Assay Guidance website for GTP binding assays
  • SPA bead technology knowledge base page

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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

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Data analysis


Visit our GTP binding data analysis page.

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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

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Custom cell lines, membranes, frozen cells, and receptors


PerkinElmer offers custom cell lines and membranes as well as custom assay development. If you are interested in a custom receptor membrane, please contact our custom teams:

ON>POINT® Custom Assay Development Services

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