| Assay Method Information | |
| | HER2 Kinase Activity Assay |
| Description: | 1) 1× kinase reaction buffer was prepared from 1 volume of 5× kinase reaction buffer and 4 volumes of water, 1 mM dithiothreitol, 5 mM magnesium chloride, 1 mM manganese chloride and 12.5 mM SEB.2) 100 nl of the diluted compound working solution was transferred into each well of a reaction plate (784075, Greiner) by an Echo 550 liquid hander. The reaction plate was sealed with a sealing film and centrifuged at 1000 g for 1 min.3) 1 ng/µL Her2 kinase solution was prepared with 1× kinase reaction buffer.4) 5 µL of the kinase solution prepared above was added to each well of the reaction plate. The plate was sealed with a sealing film, centrifuged at 1000 g for 1 min, and placed at room temperature for 10 min.5) A mixture of 2 × kinase substrate and ATP was prepared by using 1 × kinase reaction buffer, and the 2 × Her2 kinase substrate was 2 µM TK-substrate-biotin and 4 µM ATP.6) 5 µL of the mixture of 2 × TK-substrate-biotin and ATP was added to the reaction plate, centrifuged at 1000 g for 30 seconds to start the reaction.7) Her2 kinase test was performed at room temperature for 50 min of reaction.8) A mixture of Sa-XL 665 (125 nM) and TK-antibody-Cryptate was prepared by using HTRF detection buffer.9) 10 µL of the mixture of Sa-XL 665 and TK-antibody-Cryptate was added to each well, centrifuged at 1000 g for 30 seconds, and reacted at room temperature for 1 h.10) The fluorescence signals at 615 nm (Cryptate) and 665 nm (XL665) were read by Envision 2104. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |