Radioligand binding methods: practical guide and tips.
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.
Bylund
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DB
Department of Pharmacology, University of Nebraska Medical Center, Omaha 68198-6260.
Toews
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ML
D000818:Animals; D001667:Binding, Competitive; D002477:Cells / Q000378:metabolism; D007700:Kinetics; D010766:Phosphorylation; D011869:Radioligand Assay / Q000379:methods*; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000378:metabolism
D011956:Receptors, Cell Surface