SPA Assays for GTP Binding 爱游戏平台注册登录

SPA Assays for GTP Binding

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Overview
What do I need to run this assay?
SPA bead options for capturing cell membranes
³⁵S-gamma GTP radiochemicals
Protocol-in-brief
Assay optimizations
Recommended assay conditions (SPA format) for ³⁵S-gamma-GTP binding assays using 爱游戏平台注册登录 cell membrane products validated for GTP binding
Application note, posters, guides and other resources
Tips and FAQs
Data analysis
Citations
Custom cell lines, membranes, frozen cells, and receptors

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.

SPA GTP binding assay

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What do I need to run this assay?

Cell membrane expressing receptor of interest. (爱游戏平台注册登录 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 爱游戏平台注册登录 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.)

*爱游戏平台注册登录 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 爱游戏平台注册登录 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 爱游戏平台注册登录 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 coating	Bead type	Core bead type	Pack size	Catalog number
Wheat germ agglutinin (WGA)	Scintillation	PVT	100 mg	RPNQ0252
			500 mg	RPNQ0001
			25 x 500 mg	SPQ0031
			2 g	RPNQ0060
			25 x 2 g	RPNQ0063
		Ysi	250 mg	RPNQ0011
		Ysi	1 g	RPNQ0023
Wheat germ agglutinin (WGA)	Imaging	PS	50 mg	RPNQ0262
			500 mg	RPNQ0260
			25 x 500 mg	RPNQ0281
			2 g	RPNQ0308
		Yox	50 mg	RPNQ0272
			500 mg	RPNQ0270
			25 x 500 mg	RPNQ0282

SPA bead types

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³⁵S-gamma GTP radiochemicals

Two different ³⁵S-gamma GTP products are available from 爱游戏平台注册登录 , 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

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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.

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 爱游戏平台注册登录 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

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
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
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
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

爱游戏平台注册登录 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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