Assay Method Information

Assay Name:  Binding Assay
Description:  This section describes a scintillation proximity assay (SPA) to measure [3H] gabapentin ([3H]GBP) binding to membranes containing α2δ-1 and its use for profiling compounds (Calvo et al (2012) J. Biomol. Screen. 17:1041-1049).Human Cav1.2/β3/α2δ-1 Calcium Channel Membranes (Chantest) were thawed on ice, aliquotted and stored at −80° C. for subsequent use. Membranes were diluted to 200 μg/ml (3 μg/well final assay concentration (FAC)) in assay buffer (10 mM HEPES (Sigma), (pH7.4)). The [3H]GBP (Perkin Elmer) stock solution was stored at −20° C. [3H]GBP was diluted to 40 nM (10 nM FAC) in assay buffer. SPA beads (Perkin Elmer) were re-suspended at 100 mg/ml in 10 mM HEPES (pH 7.4). Beads were diluted to 40 mg/ml (0.6 mg/well FAC) in assay buffer. Nonspecific binding (NSB) was generated using an excess of pregabalin (Tocris). Pregabalin was solubilized in Milli-Q H2O at 10 mM. 10 mM pregabalin was diluted to 400 μM (100 μM FAC) in assay buffer.Compounds were diluted to 100 μM then half log diluted. These were then diluted 1:100 in assay buffer to a 4× assay concentration (1 μM FAC top dilution).SPA beads 15 μl; membranes 15 μl; pregabalin or assay buffer/test compound 15 μl and [3H]GBP 15 μl were added to a white 96 well isoplate, (Perkin Elmer). The assay plate was sealed and mixed for 10 s on a plate shaker then placed into a plate rack and slotted into the reader stacker. The plate was incubated overnight (20 hours) then read on a 1450 MicroBeta TriLux Microplate Scintillation and Luminescence Counter at ambient room temperature (RT).
Affinity data for this assay
 

If you find an error in this entry please send us an E-mail