Microplate gel-filtration method for radioligand-binding assays.
A thin-layer gel-filtration chromatographic method has been developed in a 96-well format to separate free and protein-bound ligand in radioligand-binding assays. The mobile phase in the gel-filtration plate is removed via quick centrifugation before samples are applied. Protein-bound ligand is recovered via centrifugation into another 96-well plate for radioactivity measurements. The method exhibits excellent recovery of protein-ligand complexes and less opportunity for dissociation of the complexes since it eliminates major dilution effects from the mobile phase of a column and from elution steps in conventional gel-filtration chromatography. It offers other advantages: simple, rapid, inexpensive, quantitative, and able to handle a large number of samples as required in drug discovery and clinical settings. This microplate gel-filtration method was optimized in studies of receptor-ligand interactions using estrogen receptors as examples and can be used in other radioligand-binding assays.
Liu
Jianwei
J
AstraZeneca Pharmaceuticals, 1800 Concord Pike, Wilmington, Delaware 19850, USA. jianwei.liu@astrazeneca.com
Zacco
Anna
A
Piser
Timothy M
TM
Scott
Clay W
CW
D001667:Binding, Competitive; D002498:Centrifugation; D002850:Chromatography, Gel / Q000379:methods*; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods*; D011960:Receptors, Estrogen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D013379:Substrate Specificity
D007457:Iodine Radioisotopes; D008024:Ligands; D011960:Receptors, Estrogen; D011994:Recombinant Proteins