pHSense Eu Fab Anti-Mouse IgG1, 96 wells

Assay protocol for monitoring antibody or ADC internalization

The assay begins by culturing cells in a 96-well plate. The test antibody or ADC is pre-incubated with pHSense Eu Fab at a 1:2 molar ratio in cell culture medium for 30 minutes at room temperature. The resulting complex is then added to the cells and incubated at 37°C, followed by kinetic or endpoint fluorescence measurement using a TRF-compatible plate reader.

Note: A protocol for monitoring internalization in suspension cell lines is also available. Please refer to the user manual for details.

Assay protocol for monitoring receptor and membrane protein-mediated internalization

The assay begins by culturing cells in a 96-well plate. The antibody directed against the target of interest is pre-incubated with pHSense Eu Fab at a 1:2 molar ratio in cell culture medium for 30 minutes at room temperature. This mixture is then added to the cells and incubated for 1 hour at room temperature. Following this incubation, cells are stimulated with a pharmacological compound. Fluorescence is then measured either kinetically or at endpoint using a TRF-compatible plate reader.

pHSense-based detection of GLP-1R internalization following Exendin-4 stimulation

Tag-Lite® FLAG-tagged GLP-1R cells (stable cell line, Part #: C1SU1GLP1, Revvity) were seeded in a 96-well white culture-treated plate at a density of 80,000 cells per well in complete culture medium, and incubated overnight at 37 °C with 5% CO₂. A mouse IgG1 anti-FLAG antibody and the Mouse IgG1 Negative Control (Part #: 81MOIGG1NC, Revvity) were pre-incubated at room temperature for 30 minutes with pHSense Eu Fab Anti-Mouse IgG1 at a 1:2 molar ratio in cell culture medium.

Next, 40 µL of each Fab–antibody mixture were added to the cells and incubated for 1 hour at room temperature. The GLP-1R agonist Exendin-4 was serially diluted in cell culture medium, and 10 µL of each dilution were added to the wells. After a 20-minute incubation at 37 °C, the signal was recorded using a TRF-compatible plate reader.

Results show Exendin-4 induced a dose-dependent increase in signal, with an EC₅₀ value consistent with previously reported data. No signal increase was observed when an irrelevant mouse IgG1 antibody was used as a negative control.