pHSense Eu Streptavidin, 2 x 96 Assay Points

Assay protocol for monitoring binder internalization

The assay begins with culturing cells in a 96-well plate. The biotinylated test binder is pre-incubated with pHSense™ Eu Streptavidin reagents at a 1:2 molar ratio in cell culture medium or pHSense BSA internalization buffer 1X (Part #: 81BUFBR1, Revvity) 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 measurements using a TRF-compatible plate reader.

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

Assay protocol for monitoring binder-induced receptor and membrane protein internalization

The assay begins with culturing cells in a 96-well plate. The biotinylated binder directed against the target of interest is pre-incubated with pHSense™ Eu Streptavidin at a 1:2 molar ratio in cell culture medium or pHSense BSA Internalization Buffer 1X (Part #: 81BUFBR1, Revvity) for 30 minutes at room temperature.

The mixture is then added to the cells and incubated for 1 hour at room temperature. Following incubation, cells are stimulated with a pharmacological compound.

The fluorescent signal is measured using a TRF-compatible plate reader. Readings are done repeatedly at several time-points for a kinetic study, or after appropriate incubation times for endpoint assays.

pHSense-based monitoring of anti-HER2 internalizing antibody

BT-474 cells were seeded in a 96-well white, culture-treated plate at a density of 20,000 cells per well in complete culture medium, and incubated overnight at 37°C, 5% CO₂. Biotinylated Trastuzumab (an anti-HER2 internalizing antibody) was pre-incubated at room temperature for 30 minutes with pHSense™ Eu Streptavidin at a 1:2 molar ratio in cell-culture medium. The Streptavidin-antibody mixture was then serially diluted in cell-culture medium and 50 µL of each dilution was added to the cells, followed by a 20-hour incubation at 37°C, 5% CO₂.

The fluorescent signal was recorded on an EnVision™ Nexus Multimode Microplate Reader (Part# HH36000002, Revvity).

The resulting dose-dependent increase in signal demonstrates that pHSense™ Eu Streptavidin reagents can specifically detect the internalization of the anti-HER2 antibody.