| Assay Method Information | |
| | GPR120 pERK assay |
| Description: | For the pERK assay, 3×106 cells/mL cryopreserved human and mouse cells were thawed rapidly in a 37° C. water bath and added to a T-225 flask containing 50 mL growth medium. The flasks were placed in a tissue culture incubator overnight (37° C., 5% CO2). The next day, cells were harvested with trypsin (Gibco Cat. #25300-054), resuspended in serum-containing growth medium and counted using a Cellometer and volume adjusted to a concentration of 0.5×106 cells/mL. Cells were plated into 384-well clear bottom tissue culture plates (BD Cat. #353962) at 50 μL/well, for a density of 25,000 cells/well using a MULTIDROP and incubated for 16-18 hours (overnight) at 37° C. with 5% CO2. The next day, cells were washed once with 50 μL of PBS without Ca++/Mg++ (Gibco Cat. #14190-036) and serum starved in 25 μL of F-12 media without any serum or antibiotics for 2 hours at 37° C.Test compounds were 3-fold, 11-point serially diluted in DMSO in a REMP assay plate (Matrix Cat. #4307) by Tecan and 5 μL was transferred into an ECHO source plate (Labcyte Cat. #LC-0200). Cells were then stimulated with 40 nL of compound dilutions using ECHO liquid handler for 7 min at 37° C. Compounds ranged from final assay concentrations of 32 μM to 0.54 nM.The media was then dumped and cells lysed with 20 μL of 1× Lysis buffer from the AlphaScreen SureFire Phospho-ERK 1/2 Kit (Perkin Elmer Cat. #6760617M). The lysis buffer was diluted 5-fold with water before use. The plate was agitated on a shaker for 10 min after which 2 μL was transferred into a 384-well white proxiplate (Perkin Elmer Cat. #6008289). The SureFire assay reagent mix was prepared by mixing 60 parts Reaction Buffer, 10 parts Activation Buffer, 1 part Donor Beads, 1 part Acceptor Beads (Perkin Elmer Cat. #TGRES10K). 3.5 μL/well of this reagent mix was manually added to the proxiplate with a multichannel pipettor. Plates were spun down at 1000 rpm for 2 min, followed by light-protected incubation at room temperature for 2 hours. The plates were read on the Alpha-technology compatible Envision multilabel plate reader using AlphaScreen protocol according to manufacturer's specifications. The agonist effect of compounds was expressed as 100×(average sample-average blank)/(average total-average blank) where sample is the luminescence activity in the presence of test compound, blank is equal to the luminescence activity in the presence of DMSO control and the total is signal induced by 50 μM linolenic acid as reference compound.Activation data for the test compound over a range of concentrations was plotted as percentage activation of the test compound (100%=maximum response). After correcting for background, EC50 values were determined. The EC50 is defined as the concentration of test compound which produces 50% of the maximal response and was quantified using the 4 parameter logistic equation to fit the data. |
| Affinity data for this assay | |
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