GTPγS Functional Assay Services

Creative Biolabs has been focusing on GPCR ligand-binding assay for years and is professional in radioligand binding assays. We have standardized experimental procedures to ensure the repeatability of experiments.

As GPCRs are involved in the pathophysiology of human disease, they have been considered preferential targets in drug development. Consequently, the design of biochemical assays to evaluate the coupling of GPCRs to Gα-proteins represents a wanted approach to the study of receptor functionality. The G-protein activation by selective drugs measures the functional consequence of receptor occupancy at one of the earliest receptor-mediated events. Early measurement of G-protein activation seems to be the best option to evaluate the receptor functional status as is not subjected to amplification or other modulation that may occur when analyzing parameters downstream, such as the detection of second messengers like intracellular Ca²⁺ concentration or inositol phosphate production measurement. 35-labeled guanosine-5 -O-(γ-thio)-triphosphate ([³⁵S]GTPγS) binding assay measures the level of G-protein activation following agonist occupation of a GPCR, by determining the changes in the binding of the poorly hydrolyzable GTP analog [³⁵S]GTPγS to the Gα subunit. Agonist activation of the receptor promotes the increase of the [³⁵S]GTPγS binding following a classical concentration-dependent response.

Fig.1. Receptor binding and G-protein-dependent assays. (Zhang, 2012)

Overview of GTPγS Functional Assay

The [³⁵S]GTPγS binding assay measures the level of G protein activation following agonist occupation of a GPCR. In the assay [³⁵S]GTPγS replaces endogenous GTP and binds to the Gα subunit following activation of the receptor to form a Gα-[³⁵S]GTPγS species. Since the gthiophosphate bond is resistant to hydrolysis by the GTPase of Gα, G protein is prevented from reforming as a heterotrimer and thus [³⁵S]GTPγS labeled Gα subunits accumulate and can be measured by counting the amount of [³⁵S]-label incorporated. Because the Gα subunit remains associated with the membrane this is simply done by filtering the preparation, usual membranes from cells expressing the receptor of interest, through glass-fiber filters and counting the radioactivity retained on the filters. These data can be converted to a concentration of [³⁵S]GTPγS bound/mg membrane protein, although data are often expressed as a fold or percentage increase over basal binding or a percentage of the effect produced by a known high efficacy agonist. Thus, although this assay determines the degree of binding of [³⁵S]GTPγS it is truly a functional assay because it is a consequence of the action of an activated receptor and so can be used to determine the degree of agonism and the potency of compounds acting at a particular GPCR. The classical [³⁵S]GTPγS binding technique has been used in cells, tissue membrane preparations, and autoradiography sections. This conventional [³⁵S]GTPγS binding assay is more feasible for receptors coupled to Gα_i/oproteins because of their higher relative abundance in the brain over other Gα families, and because of their higher rates of nucleotide exchange and constitutive activity.

Fig. 2. Flow chart for assay development.

Our Feature Service

• Standardized GTPγS Functional Assay Services.
• One-stop service to provide comprehensive GPCR ligand-binding assay.
• Advanced technology platform

As an industry-leading CRO company, Creative Biolabs has accumulated extensive experience during years of practice. We have optimized and standardized our experiment protocols and established a great reputation with these. If you are interested in GPCR ligand-binding assay or you have any other requirements, please don't hesitate to contact us for more information.

Reference

1. Zhang, R. and Xie, X. Tools for GPCR drug discovery. Acta Pharmacol Sin. 2012, 33(3): 372-84.