| Assay Method Information | |
| | Agonistic Activity of Polypeptide Compounds for GHSR-1a |
| Description: | The activity of GHSR-1a can be measured using different techniques, for example, by detecting the change in the intracellular conformation of GHSR, the change in G-protein coupling activity, and/or the change in intracellular messengers. Techniques such as measuring intracellular Ca2+ are preferably used to measure the activity of GHSR-1a. Examples of techniques known in the art that can be used to measure Ca2+ include the use of FLIPR calcium ion assay kits, among others. The FLIPR calcium ion assay kits use a calcium ion sensitive indicator and a masking dye to ensure that a researcher carries out high-sensitivity fluorescent screening for G protein-coupled receptors, ion channels and other calcium ion sensitive targets. This experiment used FLIPR calcium 6 assay kits and FLIPR calcium 6-QF assay kits.1. Process1.1. Cell Culture and Reagent Preparationa) Cell line: Flp In-CHO-GHSR Stable Pool;b) Complete medium: F12K+10% fetal bovine serum+1×penicillin-streptomycin (PS)+600 μg/mL hygromycin B;c) Cell seeding medium: F12K+10% fetal bovine serum.d) Assay buffer: 1× HBSS+20 mM HEPES.e) 10× component A: Assay buffer and component A were left at room temperature (RT), 10 mL of buffer was added to component A, and the mixture was vortexed for 1-2 min and stored at −20° C.1.2. Compound Managementa) Compound stock solutions: the powders from in-house synthesis were made into 10 mM stock solutions in DMSO according to the standard protocol.b) Compound storage: all compounds in DMSO were stored in room temperature desiccators for short-term storage (at most 4 months). The remaining compounds were left at −20 ° C. for long-term storage.1.3. Agonist Activity Assaya) Flp In-CHO-GHSR Stable Pool cells were cultured in complete medium.b) The cells were placed in 25 lbs/inch cell seeding medium in a 384-well cell culture plate (Corning, 3764) at 7k cells/well and cultured overnight at 37° C. with 5% CO2.c) 20× component A was thawed at room temperature, diluted with assay buffer to 2×, and left at RT.d) The Petri dish was taken out of the incubator and equilibrated at room temperature for 10 min. The medium was changed to apricot buffer. After the final wash, 20 μL of buffer was kept in each well, 20 μL of 2× component A was then added to each well, and the plate was incubated at 37° C. for 3-5 s.e) 10 μL of 5× compound was added to the 384-well cell culture plate, and data collection was immediately performed using FLIPR Tetra. |
| Affinity data for this assay | |
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