| Assay Method Information | |
| | ADORA1A Receptor Binding Assay |
| Description: | 2 L of each compound (test compounds, high control compound, low control compound) was transferred into individual wells of an assay plate. Also, to each individual well, 98 L of Adenosine A1a membrane stock (for ADORA1A Receptor Binding Assay) or Adenosine A2a membrane stock (for ADORA2A Receptor Binding Assay) was dispensed, followed by 100 L of radio-labeled ligand. Plates were then sealed and incubated at room temperature for 1 hour (for ADORA1A Receptor Binding Assay) or 2 hours (for ADORA2A Receptor Binding Assay). Unifilter-96 GF/C filter plates were pre-soaked with 50 L of 0.3% PEI per well for at least 30 min at room temperature. When binding assays were completed, reaction mixtures were filtered through GF/C plates using a Perkin Elmer Filtermate Harvester, and then each plate was washed 4 with cold wash buffer. Filter plates were then dried for 1 hour at 50 ° C. After drying, the bottom of the filter plate wells was sealed using Perkin Elmer Unifilter-96 backing seal tape. 50 L of Perkin Elmer Microscint 20 cocktail was then added, and the top of the filter plate was then sealed using Perkin Elmer TopSeal-A sealing film. 3H trapped on the filter was counted using a Perkin Elmer MicroBeta2 Reader. Data analysis was performed using GraphPad Prism 5 software, and the Inhibition [% Control] was calculated using the following equation: % Inh=(1 Background subtracted Assay value/Background subtracted HC value) *100. |
| Affinity data for this assay | |
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