| Assay Method Information | |
| | Human GPR35a Isoform Binding Assay |
| Description: | Overexpression of Human GPR35a Baculovirus in HEK293f cells at a cell density of 2.5 106 cells/mL and a multiplicity of infection of 2.5 over 24 h in Pro293 (Lonza)+5% FBS, 1% Glutamax and 0.4% Pen/Strep. Cells harvested and centrifuged at 2500 RPM for 10 mins at 4 C. The supernatant was then poured off and the pellet stored at +-80 C. The pellet was defrosted and re-suspended in 15 mL of homogenising buffer (20 mM HEPES, 10 mM EDTA, pH 7.4). Then homogenised in mechanical homogeniser (VMR) for 10 seconds. The membrane was centrifuged in centrifuge tubes at 40,000 g for 15 mins at 4 C. The supernatant was poured away and re-suspended in 15 mL of homogenising buffer. Homogenised for 20 seconds. The membrane was centrifuged at 40,000 g for 45 mins at 4 C. The membrane was re-suspended in 3 mL of storage buffer (20 mM HEPES, 0.1 mM EDTA, pH 7.4) mixing well. The resulting membranes were then stored at +-80 . GPR35 cell membrane homogenates were re-suspended in the binding buffer (50 mM TRIS+10 mM MgCl2 pH 7.4) to a final assay concentration of 5 ug/well. Test compounds were diluted in dimethylsulphoxide (DMSO (Sigma Aldrich, UK)), to form a 10 point log concentration curve. Test compounds were added per plate, followed by 7 nM 3H-27966. 0.1 uM FAC Lodoxamide was added in order to allow non-specific binding to be calculated. Finally, membrane was added to each well on the plate. After 60 min incubation at room temperature, membranes were filtered onto a unifilter, 96-well white microplate with bonded GF/B filter, pre-soaked in ddH20, with a TomTec cell harvester, and washed 5 times with distilled water. Plates were dried prior to 50 ul/well scintillant added, sealed and radioactivity measured using a MicroBeta analyser. IC50 values were derived from the inhibition curve and affinity constant (Ki) values were calculated using the Cheng-Prussoff equation, where; pKi=+-log 10 Ki. |
| Affinity data for this assay | |
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