Revvity, 81MO2EU1AB, pHSense Eu Fab Anti-Mouse IgG2, 2 x 96 wells

Overview
pHSense™ Eu Fab reagent is a covalently europium-labeled, pH-sensitive Fab fragment designed to monitor antibody or anti-drug conjugate (ADC), as well as receptor- and membrane protein-mediated internalization in real time. It binds with high affinity to the Fc region of test antibodies, particularly mouse IgG2 subtypes, and remains minimally fluorescent at neutral extracellular pH (≥7). Upon internalization, the Fab-antibody complex encounters increasingly acidic intracellular compartments such as early and late endosomes and lysosomes, where the europium signal becomes progressively stronger. This novel pH sensitive europium complex is compatible with a time-resolved fluorescence (TRF) detection, effectively eliminating most fluorescence background and significantly enhancing the signal-to-background ratio. Its unique photophysical properties enable simple and robust no-wash detection of receptor-mediated endocytosis of antibodies in plate-based assays with live cells.
How it works
pHSense Eu Fab Anti-Mouse IgG2: Internalization assay principle
pHSense Eu Fab reagent is a covalently europium-labeled, pH-sensitive Fab fragment designed to monitor antibody or anti-drug conjugate (ADC) as well as receptor- and membrane protein-mediated internalization in real time. It binds with high affinity to the Fc region of test antibodies, and remains minimally fluorescent at neutral extracellular pH (≥7). Upon internalization, the Fab-antibody complex encounters increasingly acidic intracellular compartments such as early and late endosomes and lysosomes, where the europium signal becomes progressively stronger.

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 an HTRF-compatible plate reader.

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 an HTRF-compatible plate reader. Application-Internalization
Brand-pHSense
Detection Modality-pH sensitive dye
Product Group-Fluorescent Reagent
Sample Volume-50 µL
Shipping Conditions-Shipped in Dry Ice
Target-Mouse IgG2
Target Class-Cell surface proteins, antibodies, ADCs
Target Species-Mouse
Technology-TRF
Unit Size-2 x 96 wells