Revvity, 62GTPPEC, HTRF GTP / Gi protein Binding Kit, 20,000 Assay Points

Overview
The GTP Gi binding assay detects upstream Gi protein activity at the receptor, upon GPCR stimulation in membrane extracts. This HTRF-based non-isotopic assay is simple, fast, and has a no-wash format.
The assay uses GTP gamma S and is intended for GPCR model studies, and compound pharmacological identification and characterization.
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
How it works
GTP Gi binding assay principle
The GTP Gi binding assay uses a cryptate-labelled GTP analog (donor) and  a d2-labelled anti-Gi antibody (acceptor). The GTP analog specifically binds into the GTP-binding pocket of active G proteins, while the d2-labelled antibody is specific to the Gαi subunit of G proteins. GPCR activation in presence of the two detection reagents brings the donor and acceptor into proximity, which triggers a FRET signal with an intensity proportional to the concentration of active Gi protein in the sample. GTP gamma S, a non-hydrolysable GTP analog, is used to displace the donor from the GTP-binding pocket and extinguish FRET to determine the assay non-specific signal an ensure its accuracy.

GTP Gi binding-plate assay protocol
The GTP Gi binding assay is performed in a miniaturized format using 20 µl final volume, and features a streamlined 4-step reagent addition protocol where stimulation/compounds, agonist, detection reagents, and membranes are added successively. Plates are read after 3H or ON incubation at RT, depending on the biological model. No washing steps are required.
This protocol uses optimized conditions previously determined for the biological model of interest (optimal membrane quantity/well, and supplemented Stimulation buffer #3 containing optimal concentrations of MgCL2 and GDP). The optimization procedure and recommendations are presented in the associated guides. Application-Second Messenger Detection
Automation Compatible-Yes
Brand-HTRF
Detection Modality-HTRF
Product Group-Kit
Sample Volume-10 µL
Shipping Conditions-Shipped in Dry Ice
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