Search and Browse
Download
Enter Data
|
Assay Method Information |
|
| | STING SPA Binding Assay |
| Description: | The human STING SPA binding assay measures displacement of tritium labeled 2′,3′cGAMP (cyclic (guanosine-(2′→5′)-monophosphate-adenosine-(3′→5′)-monophosphate) to biotinylated STING protein. A soluble version of recombinant STING was expressed in E. coli that lacks the four transmembrane domains and contains residues 139-379 of Q86WV6 with an R at position 232 (H232R). Based on the allele frequency of 58% of the population, H232R is considered to be a wild type (Yi, et. al., “Single Nucleotide Polymorphisms of Human STING can affect innate immune response to cyclic dinucleotides” PLOS ONE. 2013, 8(10), e77846). The STING construct has an N-terminal HIS tag, followed by a TEV protease cleavage site and an AVI tag to allow directed biotinylation by BirA biotin ligase (Beckett et al., A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. (1999) Protein Science 8, 921-929). The HIS tag is cleaved after purification and prior to biotinylation.The assay was run in 1536-well plates in a total volume of 8 μL per well by adding 8 nM [3H]-2′3′-cGAMP and 40 nM biotin-STING protein in assay buffer [25 mM HEPES (Corning 25-060-C1) pH 7.5, 150 mM NaCl (Sigma S5150), 0.5 mg/mL BSA (Gibco 15260-037), 0.001% Tween-20 (Sigma P7949), molecular grade water (Corning 46-000-CM)]. Test compounds (80 nL) were added with an acoustic dispenser (EDC Biosystems) in 100% DMSO for a final assay concentration of 1% DMSO. Plates were centrifuged for 1 min and incubated for 60 min at room temperature. Finally, (2 μL) polystyrene streptavidin SPA beads (PerkinElmer RPNQ0306) were added and plates were sealed and centrifuged for 1 min at room temperature. Plates were dark adapted for 2 h and read on a ViewLux (Perkin Elmer) for 12 min per plate. A saturation binding curve for [3H]-2′3′-cGAMP showed a KD of 3.6±0.3 nM for binding to STING, comparable to reported values for the natural ligand (Zhang et al., Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |
|
Home |
| |
Search |
| |
Deposit |
| |
SiteMap |
| |
About us |
| |
Email us |
| |
Info |
|
©2000 BindingDB. All rights reserved. |
|||||||||||||