Table 7.
Summary of Myosin Ca2+-Adenosine Triphosphatase Assay
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Compound addition to plate | 1 μL | 0.5 or 2.5 mM compound in DMSO added to 384-well plates |
| 2 | Protein addition | 44 μL | 142 nM Tris-HCl, pH7.9, containing 0.23 M KCl, 2.5 mM CaCl2 |
| 3 | Incubation time | 15 min | On ice |
| 4 | ATP addition | 5 μL | 10 mM ATP in assay buffer |
| 5 | Incubation time | 30 min | Lidded, ambient temperature |
| 6 | Malachite reagent addition | 7.5 μL | PiColorLock™ Gold mixture |
| 7 | Incubation time | 5 min | Lidded, ambient temperature |
| 8 | Stabilizer addition | 3 μL | |
| 9 | Assay Readout | 630 nm | Absorbance |
1. 0.5 or 2.5 mM compound used for 10 and 50 μM final concentrations, respectively.
8. Do not directly mix PiColorLock™ Gold with stabilizer.