| Assay Method Information | |
| | Inositol Phosphate Turnover (IP1) Assay 2 |
| Description: | The assay was performed in 384-well format. HEK cells stably expressing human GPR40 were plated at 15,000 cells per well in growth medium (DMEM/10% fetal calf serum). Cell plates were then incubated 16 hours at 37 degrees in a 5% CO2 incubator.Measurement of Inositol Phosphate Turnover (IP1) was performed using the CisBio IP-One kit (Part number 62IPAPEB). After the 16 hour incubation, the cells were washed with HEPES buffer and 10 ul of stimulation buffer (prepared as described in the kit) was added to each well. In a separate plate, compounds were diluted in DMSO (400-fold over the final concentration in the assay well) and 25 nl was acoustically transferred to the appropriate well in the assay cell plate.The plates were then incubated for 60 minutes at 37 degrees. 10 ul of detection buffer (also prepared as described in the IP-One kit) was added to each well and the plates were incubated for 60 minutes in the dark. The plates were then read in a Perkin Elmer EnVision or equivalent reader able to measure FRET. Fluorescent ratio of emission at 665 and 620 nm was then converted to IP1 concentration by back calculating from an IP1 standard curve prepared at the time of the assay. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |