Anti flag m2 hrp conjugated monoclonal antibody

Transiently transfected HEK293T cells were seeded in a poly-d-lysine-coated 96-wells plate for 24 h at 37 °C and 5% CO₂. In the case of determination of surface receptor expression levels, cells were washed with 1× PBS and fixed with 4% paraformaldehyde for 10 min. Following fixation, cells were blocked with blocking buffer (1% (w/v) BSA/PBS) for 1 h at room temperature. Afterward, cells were incubated with a 1:10,000 dilution of anti-FLAG M2 HRP-conjugated monoclonal antibody (Sigma-Aldrich) in a blocking buffer for another 0.5 h at room temperature. Then, wells were washed three times with blocking buffer and three times with 1× PBS in order. Finally, antibody binding was detected using 80 μL/well diluent SuperSignal Elisa Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). The luminance signal was measured using 800-ms intervals. Data were analyzed using GraphPad Prism version 8.0.

The transfected cells were washed with 1x PBS and fixed with 4% paraformaldehyde for 10 min. Following fixation, cells were blocked with blocking buffer (1% (w/v) BSA/PBS) for 1 h at RT. Afterward, cells were incubated with a 1:10,000 dilution of anti-FLAG M2 HRP-conjugated monoclonal antibody (Sigma-Aldrich, Catalog Number A8592, Mouse lgG1) in blocking buffer for another 0.5 h at RT. Then, wells were washed with three times blocking buffer and three times 1x PBS in order. Finally, antibody binding was detected using 80 μL/well diluent SuperSignal Elisa Femto Maximum Sensitivity Substrate (ThermoFisher Scientific). The plate was measured for luminescence (Spark Multimode microplate reader, TECAN). Finally, data were normalized to 100% of the WT PTH1R using GraphPad Prism.

Cell surface expression of the receptor subunits was detected by ELISA. Plasmids corresponding to WT and mutant ghrelin receptors were transfected as described above. After transfection, cells were re-seeded onto cell adherent reagent (Applygen) treated 96-well plates at a density of 3 × 10⁴ cells per well. Twenty-four hours later, cells were washed with PBS and fixed with 10% formaldehyde for 10 min followed by three times washing with PBS. Following fixation, cells were blocked with blocking buffer (1% BSA in PBS) for 1 h at RT. Afterward, plates were incubated with a 1:10,000 dilution of anti-FLAG M2 HRP-conjugated monoclonal antibody (SigmaAldrich) in blocking buffer for another 1 h at RT. After careful washing, 80 μL/well diluent SuperSignal Elisa Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) was added, and the luminescence was measured using a luminescence microplate reader (Tecan).