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Use of FlashPlate Technology for In Vitro Measurement of [35S]-GTPγS Binding in CHO Cells Expressing the Human 5-HT1B Receptor

J. Watson and J.V. Selkirk
SmithKline Beecham Pharmaceuticals, UK

Abstract

FlashPlate is a 96-well white polystyrene microplate with scintillant-coated wells designed for highvolume, in-plate radiobinding assays. This study investigates the use of FlashPlate as a platform for the measurement of G-protein activation by means of an assay of [35S]-GTPγS binding in Chinese hamster ovary cells. Results are compared to those obtained using a conventional filtration method.

Introduction

When an external signal is received by a membrane bound receptor, a series of cellular events takes place which results in the activation of an effector molecule. Many receptors transmit this signal via G-protein activation. This response can be used as a functional measure of receptor-ligand association.

The basic mechanism behind G-protein activation involves the exchange of bound GDP for GTP. Using a radioactive, non-hydrolyzable form of GTP, such as [35S]-GTPγS, G-protein activation can be assessed by measuring the accumulation of bound radioactivity.

We investigated the use of FlashPlate technology (Basic FlashPlate, SMP200) to measure [35S]-GTPγS binding, and briefly compared it to the conventional filtration method. The study examined 5-HT activation of human 5-HT1B receptors expressed in Chinese hamster ovary (CHO) cells.

Methods
Receptor Source

A Chinese hamster ovary cell line (ACC098) expressing the human 5-HT1B receptor was used as the receptor source. Prior to [35S]-GTPγS binding assay, the CHO cells were prepared as described by Thomas, et al ., 1995, and frozen in aliquots until required.

Filtration Assay

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