Radioligand Binding Methods for Membrane Preparations and Intact Cells
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Radioligand
Radioligand Assay
Ligand binding assay
Receptor–ligand kinetics
Radioligand binding assays are a relatively simple but extremely powerful tool for studying receptors. They allow an analysis of the interactions of hormones, neurotransmitters, growth factors, and related drugs with the receptors, studies of receptor interactions with second messenger systems, and characterization of regulatory changes in receptor number, subcellular distribution, and physiological function. As a result, these assays are widely used (and often misused) by investigators in a variety of disciplines, including pharmacology, physiology, biochemistry, immunology, and cell biology. This article presents a broad overview of the radioligand binding assay technique, primarily for the investigator who has limited experience with this technique. Practical guidelines for setting up a new assay are presented, including the receptor preparation to be used, choice of appropriate radioligand, optimizing assay conditions, and appropriate methods for data analysis. Tips for avoiding some of the common pitfalls in application of these assays are also included. The primary focus is on radioligand binding assays of membrane-bound receptors studied in membrane preparations. However, similar assay techniques can be used to study receptors on intact cells. The unique advantages and disadvantages of these intact cell binding assays are also discussed. In particular, the occurrence of regulatory changes in receptors during the course of intact cell binding assays is considered, with approaches for circumventing these complications and for using intact cell assays to advantage in studying these regulatory changes.
Radioligand
Radioligand Assay
Ligand binding assay
Cell surface receptor
Second messenger system
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Radioligand
Radioligand Assay
Ligand binding assay
Bead
Streptavidin
Magnetic bead
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AbstractRadioligand binding is an extremely powerful technique that can provide detailed information about receptor-ligand interactions both in vitro and in vivo. Several types of binding assay can be performed, including studies of the kinetics of association or dissociation of the radiolabeled ligand, and equilibrium binding experiments such as saturation and competition binding assays. Data obtained from such studies generate accurate information regarding receptor number, ligand affinity, the existence of receptor subtypes, and allosteric interactions between binding sites and/or receptors. Generally receptorligand interactions are studied using membrane preparations since these give highly reproducible results. However, the technique can also be adapted to other preparations such as whole cells, tissue slices, solubilized receptors, and even whole animals. The techniques used and problems associated with radioligand binding studies are reviewed in ref. 1.KeywordsPancreatic PolypeptideRadioligand BindingLigand AffinityRadioligand Binding StudyBinding CurfThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
Radioligand
Radioligand Assay
Receptor–ligand kinetics
Dissociation constant
Ligand binding assay
Competitive binding
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Radioligand
Radioligand Assay
Receptor–ligand kinetics
Pentazocine
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Radioligand
Ligand binding assay
Radioligand Assay
Receptor–ligand kinetics
Dissociation constant
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MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but an unlabeled reporter ligand is used instead of a radioligand. The study presented herein describes the development of MS Binding Assays that address D1 and D5 dopamine receptors. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method for the selective D1 dopamine receptor antagonist SCH23390 ((5R)-8-chloro-3-methyl-5-phenyl-1,2,4,5-tetrahydro-3-benzazepin-7-ol) was established and validated, using its 8-bromo analogue SKF83566 as an internal standard. This quantification method proved to be suitable for the characterization of SCH23390 binding to human D1 and D5 receptors. Following the concept of MS Binding Assays, saturation experiments for D1 and D5 receptors were performed, as well as competition experiments for D1 receptors. The results obtained are in good agreement with results from radioligand binding assays and therefore indicate that the established MS Binding Assays addressing D1 and D5 receptors are well-suited substitutes for radioligand binding assays, the technique that has so far dominated affinity determinations toward these targets.
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Radioligand Assay
Ligand binding assay
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Radioligand binding has been used for many years to identify new binding sites, characterize receptors, and identify novel ligands. Although various techniques have been developed to improve the efficiency of preparing the biological source of the receptors and for detecting bound radioligand, the principles of the assays remain the same. This unit reviews theory and provides examples of the parameters that can be calculated from radioligand binding data to characterize ligand-receptor interactions. The important aspects of assay development and validation that allow meaningful interpretation are discussed. The selection of a radioligand, buffer and other assay components is critical to developing a useful binding assay. The nature of the binding interaction can also be probed by varying assay conditions.
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Ligand binding assay
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Radioligand
Dihydroalprenolol
Dissociation constant
Radioligand Assay
Scatchard plot
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Background and Purpose Ligand–receptor binding kinetics is receiving increasing attention in the drug research community. The Motulsky and Mahan model, a one‐state model, offers a method for measuring the binding kinetics of an unlabelled ligand, with the assumption that the labelled ligand has no preference while binding to distinct states or conformations of a drug target. As such, the one‐state model is not applicable if the radioligand displays biphasic binding kinetics to the receptor. Experimental Approach We extended the Motulsky and Mahan model to a two‐state model, in which the kinetics of the unlabelled competitor binding to different receptor states (R 1 and R 2 ) can be measured. With this extended model, we determined the binding kinetics of unlabelled N ‐5′‐ethylcarboxamidoadenosine (NECA), a representative agonist for the adenosine A 1 receptor. Subsequently, an application of the model was exemplified by measuring the binding kinetics of other A 1 receptor ligands. In addition, limitations of the model were investigated as well. Key Results The kinetic rate constants of unlabelled NECA were comparable with the results of kinetic radioligand binding assays in which [ 3 H]‐NECA was used. The model was further validated by good correlation between simulated results and the experimental data. Conclusion The two‐state model is sufficient to analyse the binding kinetics of an unlabelled ligand, when a radioligand shows biphasic association characteristics. We expect this two‐state model to have general applicability for other targets as well.
Radioligand
Receptor–ligand kinetics
Radioligand Assay
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