Description
The Glucagon assay offers superior sensitivity and ease-of-use for a reliable quantification of glucagon in cell culture media. Built on a truly homogeneous protocol, the assay does not require any washing or complex extraction steps, meaning significant time savings over ELISA methods. Simply add the detection reagents, incubate, and read!
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Assay principle
Glucagon is measured using a sandwich immunoassay with two monoclonal antibodies, one labelled with Lumi4-Tb Cryptate (donor) and the other with d2 (acceptor). The intensity of the FRET signal obtained is proportional to the concentration of glucagon in the sample.
Assay protocol
10 µL of cell supernatants are supplemented with 5 µL of acceptor-labelled antibodies and 5 µL of donor-labelled antibodies (or 10 µL of pre-mixed reagents for a single dispensing step). The mix is then incubated from 4h to overnight at RT and results are read on an HTRF compatible reader.
Validation on mouse isolated pancreatic islets
15 pancreatic islets isolated from a C57BL/6J mouse were exposed to increasing concentrations of Glucose or Adrenaline. Following exposure, supernatants were collected and their Glucagon levels quantified. In the presence of glucose, Glucagon levels were readily detected using as few as 15 islets per well. All results were similar to those previously published by other investigators. Courtesy of Physiopathology of pancreatic BETA cell research team, Institute de Génomique Fonctionnelle, Montpellier, France.
