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| | Radioligand Competition Binding Assay |
| Description: | For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer). |
| Affinity data for this assay | |
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