| Assay Method Information | |
| | [35S]-GTPγS Binding Assay |
| Description: | CHO-hCX3CR1 membranes (5 μg per well) together with different concentrations of antagonistic compound were incubated in 50 mM HEPES (pH 7.4), 100 mM NaCl, 5 mM MgCl2, 10 μM GDP and 0.01% gelatin in a MicroWell 96-well plate (Nunc). 0.56 μCi ml-1 [35S]-GTPγS and EC80 of CX3CL1 were then added. The plate was incubated at 30° C. for 1 h and subsequently unbound [35S]-GTPγS was separated from bound by vacuum filtration to a Printed Filtermat B (PerkinElmer) using a Skatron Micro96 harvester and dried at 50° C. for 1 h. The filters were soaked with a melt-on scintillator sheet (MeltiLex, PerkinElmer), sealed using a MeltiLex heat sealer and measured in a MicroBeta Trilux reader (PerkinElmer). The different antagonistic compound concentrations were achieved by stepwise dilution in DMSO to achieve a final DMSO concentration of 1% in all wells after addition of assay buffer, regardless of compound concentration. |
| Affinity data for this assay | |
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