AlphaScreen Anti-GST Donor Beads, 1 mg

Principle of the Alpha Anti-GST Donor Beads detection

AlphaLISA is a bead-based assay technology used to study biomolecular interactions in a microplate format. AlphaLISA assays require two bead types: Donor beads and Acceptor beads. Protein-protein interaction brings the Donor and Acceptor beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor bead, enabling the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the binding of the two partners. In the example shown here, Alpha Anti-GST Donor bead binds to the GST tag, while tagged partner B* binds to an Anti-Tag antibody conjugated to an AlphaLISA, AlphaScreen® or AlphaPlex™ Acceptor bead.

*Partner B can also be biotinylated, 6xHis-tagged, or unlabeled. In these cases, use the corresponding Alpha reagent (i.e Streptavidin, anti-6xHis, protein A, …) conjugated to an Acceptor bead for the detection.

Assay protocol of the Alpha Anti-GST Donor Beads detection

The example describes the protocol using a 20 µL final assay volume for the detection of an interaction between a GST-tagged Partner A and a tagged Partner B* in a presence of a sample (e.g. inhibitor). Dispense Sample (5 µL), add the two partners (10 µL), and a mix of Alpha Anti-GST donor beads and Anti-Tag antibody conjugated to AlphaLISA, AlphaScreen or AlphaPlex  Acceptor beads (5 µL), then incubate and read.

*Partner B can also be biotinylated, 6xHis-tagged, or unlabeled. In these cases, use the corresponding Alpha reagent (i.e Streptavidin, anti-6xHis, protein A, …) conjugated to an Acceptor bead for the detection.

Validation of Alpha Anti-GST Donor Beads in a protein-protein interaction detection

The Alpha Anti-GST Donor Beads were validated for detecting protein-protein interactions (PPI). In this case study, we assessed the effect of three compounds (BI-2852, BAY-293, and MRTX-1257) on the binding of KRAS G12C and SOS1. The PPI assay was conducted in an AlphaPlate-384, Shallow Well (#6008350). We added 2 µL of the sample and 4 µL of 6xHis-tagged SOS1 protein (1 nM final), incubated for 1 hour at 23°C, then added 4 µL of a freshly prepared mix of GST-tagged KRAS G12C protein (20 nM final) and GTP (10 µM final), and 10 µL of a freshly prepared mix of AlphaLISA Anti-6xHis Acceptor beads (#AL178C/M/R) (20 µg/mL final) and Alpha Anti-GST Donor beads (20 µg/mL final). Detection reagents were prepared in AlphaLISA PPI buffer (#AL015C/F). After a 2-hour incubation at 23°C in the dark, the Alpha signal was recorded using the Envision Nexus reader. As expected, all three compounds induced a dose-dependent decrease in the binding of KRAS G12C and SOS1.

Validation of Alpha Anti-GST Donor Beads for protein-ligand interaction detection

The Alpha Anti-GST Donor Beads were validated for detecting protein-ligand interactions. In this case study, we assessed the effect of three compounds (KI696, ML334, and Bardoxolone) on the binding of human Keap1 (Kelch-like ECH-associated protein 1) and a biotinylated ligand. The binding assay was performed in a 384-well shallow plate by adding 5 µL of the sample, 5 µL of GST-tagged Keap1 protein (5 nM final), 5 µL of biotinylated ligand (0.6 nM final), and 5 µL of a freshly prepared mix of AlphaLISA Streptavidin Acceptor beads (#AL125C/M/R) (20 µg/mL final) and Alpha Anti-GST Donor beads (20 µg/mL final). Detection reagents were prepared in AlphaLISA Binding Assay Buffer (#AL018C/F). After an overnight incubation at 23°C in the dark, the Alpha signal was recorded using the Envision Nexus reader.

As expected, the two orthosteric KEAP1 compounds, KI696 and ML334, induced a dose-dependent decrease in the binding of Keap1 and the biotinylated ligand. In contrast, Bardoxolone, an allosteric KEAP1 compound, had no effect on this binding.