Description
pHSense Eu Anti-HA is non cell permeant, and labeled with a pH sensitive europium complex. It is specifically designed to monitor the internalization of receptors and membrane proteins tagged with an HA epitope on their extracellular domain. This mouse monoclonal anti-HA antibody (IgG2b) is labeled with pHSense and raised against the twelve-amino-acid peptide sequence CYPYDVPDYASL. It recognizes the influenza virus hemagglutinin epitope (YPYDVPDYA), which has been used extensively as a general epitope tag in expression vectors. At neutral extracellular pH (≥7), its fluorescence remains minimal. Once internalized, the pHSense Eu Anti-HA encounters increasingly acidic compartments such as early and late endosomes and lysosomes, where the europium signal becomes progressively stronger. This new pH sensitive europium anti-HA 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 in plate-based assays with live cells.
pHSense Eu-Anti-HA assay principle
pHSense Eu Anti-HA is non cell permeant, and labeled with a pH sensitive europium complex. This Monoclonal Anti-HA antibody is designed to monitor receptor and membrane protein internalization when tagged with an HA epitope. The antibody recognizes the HA sequence and remains minimally fluorescent at neutral extracellular pH (≥7). Once internalized, the pHSense Eu Anti-HA encounters increasingly acidic compartments such as early and late endosomes and lysosomes, where the europium fluorescent signal becomes progressively stronger.
pHSense Eu Anti-HA assay protocol
The assay begins with culturing cells in a 96-well plate. The pHSense Eu Anti-HA 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 GLP1R internalization following exendin-4 stimulation
HEK-293 cells were plated in a 96-well white, culture-treated plate and transfected with a GLP1R-HA-Tagged plasmid.
After cell supernatant removal, 40 µL of the pHSense Eu Anti-HA diluted in cell culture medium (DMEM +10%FBS) were added to the cells, followed by a 1 hour incubation at room temperature.
Exendin-4, a GLP1R agonist, was serially diluted in cell culture medium, and 10 µL of each dilution were added to the wells. After incubation at 37°C, the signal was recorded using a TRF-compatible plate reader. As expected, Exendin-4 induced a dose-dependent increase in signal, with an EC50 value consistent with previously reported data.
