Gβγ-GIRK1 Interaction Assay www.perkinelmer.com A L P H A S C R E E N A P P L I C AT I O N N O T E A N 0 0 5 - A S C Application Note Introduction AlphaScreen™ Gßγ-GIRK1 Interaction is a fully homogeneous assay in 384-well format, designed to measure interaction between G protein ßγ subunit and the carboxyl terminal domain of the GIRK1 potassium channel (named cGIRK1). Gßγ is biotinylated and binds to streptavidin-donor beads, while cGIRK1 binds to anti GST-acceptor beads via its GST tag. Specific interaction between the two proteins leads to signal increase. The binding is fully reversible and completely abolished by competition with G protein α subunit. This assay can be easily applied to any protein-protein interaction. biotin-Gβγ + cGIRK1-GST + Gα Gαβγ biotin-Gβγ / cGIRK1-GST cGIRK1-GST 680 nm Gβγ 520-620 nm Anti-GST conjugated acceptor bead Streptavidin-coated donor bead 2 Example #1: Competition of biotin-Gβγ binding to GST-cGIRK1 by Gβγ Assay developed in PerkinElmer 384-well OptiPlate™ Reagents 1. Biotinylated Gβ1γ2 (12.3 µM in 20 mM Hepes, 100 mM NaCl, 0.1% CHAPS): dilute to 125 nM with assay buffer 2. GST-cGIRK1 (14.7 µM in 25 mM Hepes pH 8.0, 50 mM NaCl, 5% glycerol): dilute to 50 nM with assay buffer 3. Gβ1γ2 (22.1 µM in 20 mM Hepes, 100 mM NaCl, 0.7% CHAPS): dilute to 30 µM - 5 nM with assay buffer 4. Streptavidin-donor beads (5 mg/ml in 25 mM Hepes,pH 7.4): dilute to 50 µg/ml with assay buffer 5. Biotin-Gβ1γ2 / streptavidin-donor bead complex: mix equal volumes of biotinylated Gβ1γ2 and streptavidin-donor beads and incubate for 30 minutes at RT 6. Anti GST-acceptor beads (5 mg/ml in 25 mM Hepes, pH 7.4): dilute to 125 µg/ml with assay buffer Assay buffer: 100 mM Hepes, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 0.1% BSA Protocol: 1. Add 5 µl GST-cGIRK1. 2. Add 5 µl Gβ1γ2. 3. Add 5 µl anti GST-acceptor beads. 4. Incubate 30 minutes at RT. 5. Add 10 µl biotin-Gß1γ2 / streptavidin-donor bead complex. 6. Incubate 60 minutes at RT. 7. Read plate. 3 Example #2: Inhibition of biotin-Gβγ binding to GST-cGIRK1 by Gαi Assay developed in PerkinElmer 384-well OptiPlate™ Reagents 1. Biotinylated Gβ1γ2 (12.3 µM in 20 mM Hepes, 100 mM NaCl, 0.1% CHAPS): dilute to 125 nM with assay buffer 2. GST-cGIRK1 (14.7 µM in 25 mM Hepes pH 8.0, 50 mM NaCl, 5% glycerol): dilute to 50 nM with assay buffer 3. Gαi1 (11.5 µM in 20 mM Hepes, 100 mM NaCl, 0.7% CHAPS): dilute to 3 µM with assay buffer, incubate for 2h at 30°C in 25 mM Tris-HCl, pH 7.5, 0.5 mM MgCl2, 100 µM GDP, then further dilute to 2.5 µM - 5 nM with assay buffer 4. Streptavidin-donor beads (5 mg/ml in 25 mM Hepes, pH 7.4): dilute to 50 µg/ml with assay buffer 5. Anti GST-acceptor beads (5 mg/ml in 25 mM Hepes, pH 7.4): dilute to 125 µg/ml with assay buffer 6. GST-cGIRK1 / anti GST-acceptor beads complex: mix equal volumes of GST-cGIRK1 and antiGST-acceptor beads and incubate for 30 minutes at RT Assay buffer: 100 mM Hepes, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 0.1% BSA Protocol: 1. Add 5 µl biotin-Gß1γ2. 2. Add 5 µl Gαi1. 3. Add 5 µl streptavidin-donor beads. 4. Incubate 30 minutes at RT. 5. Add 10 µl GST-cGIRK1 / antiGST-acceptor bead complex. 6. Incubate 60 minutes at RT. 7. Read plate. 4 Example #3: Reversibility of biotin-Gβγ / GST-cGIRK1 interaction Assay developed in PerkinElmer 384-well OptiPlate™ Reagents 1. Biotinylated Gβ1γ2 (12.3 µM in 20 mM Hepes, 100 mM NaCl, 0.1% CHAPS): dilute to 125 nM with assay buffer 2. GST-cGIRK1 (14.7µM in 25 mM Hepes pH 8.0, 50 mM NaCl, 5% glycerol): dilute to 50 nM with assay buffer 3. Gβ1γ2 (22.1 µM in 20 mM Hepes, 100 mM NaCl, 0.7% CHAPS): dilute to 5 µM with assay buffer 4. Streptavidin-donor beads (5 mg/ml in 25 mM Hepes, pH 7.4): dilute to 50 µg/ml with assay buffer 5. Anti GST-acceptor beads (5 mg/ml in 25 mM Hepes, pH 7.4): dilute to 125 µg/ml with assay buffer Assay buffer: 100 mM Hepes, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 0.1% BSA Protocol for binding association: 1. Add 5 µl GST-cGIRK1. 2. Add 5 µl anti GST-acceptor beads. 3. Incubate 30 minutes at RT. 4. Add 5 µl biotin-Gβ1γ2. 5. Incubate 30 minutes at RT. 6. Add 10 µl streptavidin-donor beads. 7. Incubate 10 minutes at RT. 8. Read plate. Protocol for binding dissociation: 1. Add 5 µl GST-cGIRK1. 2. Add 5 µl biotin-Gβ1γ2. 3. Add 5 µl anti-GST-acceptor beads. 4. Incubate 30 minutes at RT. 5. Add 5 µl Gβ1γ2. 6. Incubate 10 minutes at RT. 7. Add 5 µl streptavidin-donor beads. 8. Incubate 10 minutes at RT. 9. Read plate. 5 PerkinElmer Life and Analytical Sciences, 710 Bridgeport Avenue, Shelton, CT 06484 USA (800) 762-4000 or (+1) 203-925-4602 PerkinElmer Life and Analytical Sciences, Imperiastraat 8, BE-1930 Zaventem Belgium Technical Support: in Europe: [email protected] in US and Rest of World: [email protected] Belgium: Tel: 0800 94 540 • France: Tel: 0800 90 77 62 • Netherlands: Tel: 0800 02 23 042 • Germany: Tel: 0800 1 81 00 32 • United Kingdom: Tel: 0800 89 60 46 Switzerland: Tel: 0800 55 50 27 • Italy: Tel: 800 79 03 10 • Sweden: Tel: 020 79 07 35 • Norway: Tel: 800 11 947 • Denmark: Tel: 80 88 3477 • Spain: Tel: 900 973 255 © 2003 PerkinElmer, Inc. All rights reserved. PerkinElmer is a registered trademark and AlphaScreen and OptiPlate are trademarks of PerkinElmer, Inc. PerkinElmer reserves the right to change this brochure at any time without notice and disclaims liability for editorial, pictorial, or typographical errors. Printed in USA. 006815 [M3337 6/00] www.perkinelmer.com