Solution Informationhelp
Enzyme: G-protein coupled receptor 35
inhibitor: BDBM64829
substrate: n/a
 
Solution Type: Aqueous
pH at Preparation: n/a
Temp. Prep.: n/a
Comments: Assay Materials: 1) 96-well plates (FALCON 353075) 2) U2OS (Human Osteosarcoma) cell line stably expressing the Beta-arrestin GFP and human GPR35 receptor 3) Culture Media: DMEM with 10% Fetal Bovine Serum and selection antibiotics - 200ug/ml G418 and 100ug/ml Zeocin 4) EC80 concentration of Agonist: 10 uM Zaprinast 5) DMSO solution 6) Test compounds Working Solution: 10mM Stock in 100% DMSO, diluted in assay buffer (HBSS Cellgro #21-023-CV) 7) Fixative Solution: 4% Paraformaldehyde (PFA) diluted in PBS 8) Permeabilization Solution: 0.1% Triton X-100 in PBS 9) Primary phosphor-ERK1/2 antibody (Cell Signaling Technology, diluted 1:100) 10) Goat anti-rabbit 800CW secondary antibody diluted 1:800 in Licor blocking buffer together with Sapphire700 (diluted 1:1000) and DRAQ5 (diluted 1:2000) for normalization purpose Assay Procedure: 1) Cells were grown to confluence in 96-well plates and serum-starved overnight prior to assay. 2) Prior to drug treatment cells were washed once with HB
 
 
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