Description
This plasmid coding for beta-arrestin 2 is intended as a companion product to the HTRF beta-arrestin 2 recruitment kit (62BDBAR2PEB/C) and to the HTRF Total Beta-arrestin 2 cellular kit (64BAR2TPEB/C). It enables the overexpression of beta-arrestin 2 in cell lines that have low endogenous levels of it, and helps to improve the resulting assay window. The use of this plasmid is especially advised in CHO cell lines that can otherwise prove difficult to use in beta-arrestin recruitment assays due to their low expression of the protein.
Tag-lite assays offer many features and applications over traditional receptor/ligand binding assays:
Non-radioactive
Homogeneous and filtration-free
Ready-to-use kits and reagents for binding assays
Does not alter the receptor pharmacology
Peer-reviewed, validated technology
Thorough kinetic studies with true Kd and Ki values, association (Kon) and dissociation (Koff) rate constants
Screening and profiling of biologics and large molecules
HTRF WT Beta-arrestin plasmid transfection principle
Stable CHO or HEK293 cells expressing GPCR are plated at 80,000 cells/well in a 96-well plate, and incubated for 24h at 37°C, 5% CO2.
The medium is then removed, and 50µL of a mixture of lipofectamin + DNA in optiMEM without SVF are added to the well. It is strongly advised to test 3 different amounts of beta-arrestin 2 plasmid to ensure identification of the best performances of the Beta-arrestin2 recruitment kit, e.g. 5, 10, & 20 ng of beta-arrestin 2 plasmid.
After 6 h of incubation at 37°C, 5% CO2, 100µL of medium with SVF are added to the well.
After 24h at 37°C, 5% CO2, the cells are ready to run various assays.
We recommend working with two different plates for the transfection process. The first plate is used to run the Beta-arrestin 2 recruitment assay on the cells treated with increasing concentrations of compounds (assessment of the assay window and EC50 for the different plasmid quantities). The second plate is used to run the HTRF Total Beta-arrestin 2 and HTRF Total AP2 assays, and assess beta-arrestin 2 and AP2 expression levels post transfection.
