S]-GTPγS binding was carried out as described by Watson, et al ., 1996. In brief, 108 cells were pre-incubated at 30C for 30 minutes in HEPES [20 mM], pH 7.4, in the presence of MgCl2 [3 mM], NaCl [100 mM], ascorbate [0.2 mM] and GDP [10 mM] with or without test drugs. The reaction was started by adding 10 μl of [35
S]-GTPγS [100 pM]. This was followed by a further incubation at 30C for 30 minutes. The reaction was stopped by rapid filtration, followed by five washes with ice-cold HEPES buffer. Bound radioactivity was determined by liquid scintillation spectrometry. Non-specific binding was determined by 10 μM unlabeled GTPγS.
Membrane immobilization onto FlashPlate
CHO cells expressing the h5-HT1B receptor were diluted to a desired membrane protein concentration in 25 mM HEPES buffer containing 2.5 mM CaCl2 and 1 mM MgCl2, at pH 7.4. A suspension of diluted receptor (230 μl) was pipetted into each well of a FlashPlate microplate, and the plates were centrifuged at 800 x g for 10 minutes at 4C. The supernatant was removed and the plates were left overnight at 4C. (Later studies have shown that 3 hours is sufficient.) Prior to use, 250 μl of 0.5% BSA in the above HEPES buffer was added to each well and left for 30 minutes at room temperature, in order to block nonspecific binding sites.
Binding Assay on FlashPlate
This method is essentially similar to the filtration method, with some modifications. The same assay buffer was used for both methods.
50 μl of test drug added to appropriate well
50 μl of assay buffer (basal) or 50 μl of GTPγS (NSB) added to appropriate well
50 μl of GDP added to all wells
100 μl of buffer added to all wells
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