Bioanalysis Group, Neuropsychopharmacology Program, Municipal Institute for Medical Research-Hospital del Mar, 08003 Barcelona, Spain.
Anal Biochem. 2010 Apr 15;399(2):174-81. doi: 10.1016/j.ab.2010.01.003. Epub 2010 Jan 11.
Receptor binding techniques have been widely used in different biochemical applications, with isolated membranes being the most used receptor preparation in this type of assays. In this study, intact cells were compared with isolated membranes as receptor support for radioligand receptor binding assay. The growth hormone secretagogue receptor 1a (GHSR-1a) expressed in human embryonic kidney 293 (HEK293) cells was used as a model of G-protein-coupled receptors. Differences between using intact cells in suspension and using isolated membranes were evaluated for different aspects of the receptor binding assay: total binding variations while both receptor preparations remain on ice, modifications in incubation conditions, saturation, and competition using different agonists. Intact cells are more prone to variability. Although under optimized settings both preparations were equivalent, the K(d) value for intact cells was three times higher than that using isolated membranes. However, no significant differences were observed in competition assays obtaining practically identical K(i) values for all ligands tested. For the GHSR-1a, isolated membranes are the better choice if particular incubation conditions are required (less variability), whereas intact cells yield easy, fast, and physiological conditions for receptor binding assays.
受体结合技术已广泛应用于不同的生化应用中，其中分离的膜是此类测定中最常用的受体制备方法。在这项研究中，将完整细胞与分离的膜进行了比较，作为放射性配体受体结合测定的受体支持。生长激素促分泌素受体 1a（GHSR-1a）在人胚肾 293（HEK293）细胞中表达，用作 G 蛋白偶联受体的模型。评估了悬浮完整细胞和分离膜在受体结合测定的不同方面的差异：两种受体制剂仍在冰上时的总结合变化、孵育条件的改变、使用不同激动剂的饱和和竞争。完整细胞更容易发生变化。尽管在优化的条件下，两种制剂是等效的，但完整细胞的 K(d)值是使用分离膜的三倍。然而，在竞争测定中没有观察到显著差异，所有测试配体的 K(i)值几乎相同。对于 GHSR-1a，如果需要特定的孵育条件（变异性较小），则分离的膜是更好的选择，而完整的细胞则为受体结合测定提供了简单、快速和生理的条件。
