Radioligand binding methods: practical guide and tips

Abstract

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.

GTP Binding and Signaling by Gh/Transglutaminase II Involves Distinct Residues in a Unique GTP-binding Pocket

“…3B) indicated that, in addition to G h /fXIIIA and S171E, K173L and K173N were impaired in GTP binding. This is informative, because in our hands, WT G h has an affinity for GTP of ϳ25 nM, 2 and affinities lower than this cannot be readily detected by filtration assays (28). Thus, the lack of GTP␥S binding by K173L and K173N, both of which photolabel, indicates that these two mutants have a lower affinity for GTP than WT.…”

Identification of α-1L Adrenoceptor in Rabbit Ear Artery

“…␣-1L ARs could not be detected in the membrane preparations, even though the binding conditions were identical to those used for the intact tissue segment binding procedures. The classical radioligand binding assay using microsomal membrane preparations eliminates some nonspecific binding (Bylund and Toews, 1993;Mackenzie et al, 2000) but may result in a loss of receptors upon homogenization and fractionation . This type of loss of receptor upon homogenization has been suggested to be especially important for small tissues (Colucci et al, 1981;Faber et al, 2001).…”

“…However, a selective loss of ␣-1L AR, in contrast with the ␣-1A AR, cannot be ruled out. Since tissue homogenization changes receptor environment, binding to receptors in membrane preparations may not reflect the binding to receptors present in intact cells (Bylund and Toews, 1993). It is possible that a selective loss of the ␣-1L AR binding phenotype is related to a conformational modification or the environmental changes caused by homogenization.…”

“…However, it has been pointed out that tissue homogenization may cause dramatic changes in the membrane receptor environment, possibly leading to changes in receptor binding properties (Bylund and Toews, 1993). Recently, we have reevaluated the use of intact tissue segments for measurements of ligand binding, because intact tissue may reflect better the physiological state of a receptor.…”

Synthesis of Sulochrin-¹²⁵I and Its Binding Affinity as α-Glucosidase Inhibitor using Radioligand Binding Assay (RBA) Method

“…The low value of this linearity of curve ( r ) is might be due to less optimal separation of bound radioligand from free radioligand. The separation is crucial step in RBA method, it is important to prevent dissociation of the receptor-radioligand complex which this parameter is measured [17]. The value of Kd and Bmax was obtained from this equation i.e Kd = -(1/slope) = -(1/-0.038) = 26.316 nM and Bmax = intercept = 9.302 nM.…”