Revvity, 62CGTPPEC, HTRF Cellular GTP / Gi protein Binding Kit, 20,000 assay points

Overview
This cellular GTP / Gi binding assay is a fluorescent and homogeneous format to measure Gi protein activity in cells at the level of the GDP/GTP nucleotide exchange that characterizes GPCR activation. As such, it has the advantage of monitoring the activation of GPCRs at the level of one of the earliest receptor-mediated events. It therefore provides insight into the upstream portion of GPCR signaling, useful when studying functional responses.
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 readout time or size of assays
How it works
GTP Gi cellular binding assay principle
The GTP/Gi cellular binding assay is intended for the simple, rapid, and direct detection of Gαi protein activation in permeabilized cells. It is an upstream readout of Gi protein coupled receptor activation. GPCR activation leads to GDP/GTP nucleotide exchange into the Gα subunit. The principle of this assay is based on HTRF™ technology. It uses a non-hydrolysable GTP analog coupled to the fluorescent Europium cryptate donor.
In practice, agonist-induced GPCR stimulation leads to Gα protein conformation change, and the replacement of Gα-bound GDP by the fluorescent GTP analog in the corresponding binding pocket. Detection is made possible by the addition of d2-labeled anti-Gαi monoclonal antibody (red acceptor). When Europium cryptate and d2 are brought into close proximity, the time-resolved energy transfer between them triggers a TR-FRET signal at d2. This specific signal is proportional to the Gαi activation state. The assay enables the direct pharmacological characterization of compounds acting on Gαi-coupled receptors in permeabilized cells.

 
GTP Gi cellular binding assay protocol
The GTP/Gi cellular binding assay is performed in a miniaturized format using 40 µl final volume and features a streamlined 4-step reagent addition protocol where permeabilization, stimulation, and detection steps are performed successively after plating cells for 24h. Plates are read after 4H or ON incubation at RT, depending on the biological model.
Optimal cell densities, MgCl2 concentrations, and incubation times before reading must be determined based on the chosen biological model (cellular model/receptor/pharmacological compound). Application-Second Messenger Detection
Brand-HTRF
Detection Modality-HTRF
Product Group-Kit
Shipping Conditions-Shipped in Dry Ice
Target-Gi protein
Target Class-GPCR
Technology-TR-FRET
Therapeutic Area-Cardiovascular Infectious Diseases Inflammation Metabolism/Diabetes NASH/Fibrosis Neuroscience Oncology & Inflammation Rare Diseases
Unit Size-20,000 assay points