AlphaScreen experiments were performed at 25 °C in the incubation buffer. 15 μL of the reaction mixture containing the incubation buffer, 5 nM SlCOI1, 10 nM Fl-SlJAZPs and various concentrations of COR or JA-Ile was added to a 1/2 Area AlphaPlate-96 (PerkinElmer), and then incubated for 1 h at 4 °C. Then, 10 μL of a detection mixture containing incubation buffer, 0.1 μL of FITC-coated donor beads, 0.1 μL of GST-coated acceptor beads was added to each well. Finally, the mixture was incubated for 12 h and the luminescence signals were detected using the Envision 2105 Multimode Plate Reader (PerkinElmer). The experiment was repeated three times, and the data are presented as average values with standard deviation. To clarify the difference in the affinity of the ligands with each COI1/JAZ co-receptor, the results of the AlphaScreen assay are shown with the normalized signal intensity change (Figs. 3 , 4 , and Supplementary Fig. S9 ) along with the raw signal intensity (Supplementary Figs. S8 , S10 , and S13 )40 (link).
Alphaplate 96
Manufactured by PerkinElmer
The AlphaPlate-96 is a 96-well microplate designed for high-throughput screening and detection applications. It features a proprietary surface that enables efficient, homogeneous assay performance with a variety of AlphaLISA and AlphaScreen assays.
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Lab products found in correlation
2 protocols using alphaplate 96
1
Quantifying Ligand-COI1/JAZ Interactions
2
AlphaScreen Protein-Protein Interaction Assay
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AlphaScreen experiments were performed at 25 °C in the incubation buffer (50 mM Mops, pH 7.4, 50 mM NaF, 50 mM CHAPS, 0.1 mg/ml bovine serum albumin) (31 (link)). 15 μl of the reaction mixture in the incubation buffer containing 50 nM FLAG-MYC and 150 nM GST-Bls-MED25 (or GST-Bls-JAZ9) was added to a 1/2 Area AlphaPlate-96 (PerkinElmer) and incubated for 2 h at 4 °C. Subsequently, 10 μl of a detection mixture in the incubation buffer containing 0.1 μl of anti-FLAG antibody-coated AlphaScreen acceptor beads and 0.1 μl of Streptavidin-coated donor beads was added to each well. In Figure S13 B, anti-FITC antibody-coated donor beads were used instead of Streptavidin-coated donor beads. Thereafter, the mixture was incubated for 5 h at room temperature, and the luminescence signals were detected using the Envision 2105 Multimode Plate Reader (PerkinElmer, Inc.). The experiment was repeated three times, and the data are presented as average values with SD.
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