| Assay Method Information | |
| | Biochemical Assay |
| Description: | In our experiments, bovine skinned cardiac myofibrils were isolated from the frozen bovine left ventricle as myosin's source in the ATPase assay. The calcium concentration that achieves a 50% (pCa50 or pCa=6.25) activation of the myofibril system was chosen as the final condition for assessing the activation activity according to the literature (DOI: 10.1074/jbc.M117.776815). Myofibrils ATPase activity was measured in a buffered solution containing 12 mM PIPES (piperazine-N, N′-bis(2-ethane sulfonic acid) and 2 mM magnesium chloride at pH 6.8 (PM12 buffer). Final assay conditions were 1 mg/mL of bovine cardiac myofibrils, 1:20 of stock PK/LDH (Sigma-Aldrich, Cat No. P0294-5X5ML), 50 μM ATP, 1 mM DTT (dithiothreitol), 0.75 mM NADH, 1.5 mM PEP at pCa50 (pCa=6.25). Compounds were dissolved in DMSO (dimethyl sulfoxide). Serial dilution of compounds was created such that the final desired concentration of compound would be achieved in a volume of 150 μL with a fixed DMSO concentration of 2% (v/v). 75 μL of a solution containing bovine cardiac myofibrils, PK/LDH, and calcium were added to a 96 well plate for a 7 point dose-response. In some circumstances, 10 point-response was used to repeat the ATPase assays on compounds of interest. Compounds were added to the myofibrils solution and incubated for 5 minutes. The enzymatic reaction was started with the addition of 75 μL of a solution containing ATP, PEP, NADH, compounds, and calcium. The ATPase activity was measured by reading absorbance at 340 nm in a PerkinElmer Victor Nivo plate reader at 25° C. in kinetic mode for 15 minutes using clear bottom plates. The slopes of the absorbance changes as a function of time for the first 10 minutes were normalized to slopes on the control wells containing all reagents, including DMSO, but without compounds. |
| Affinity data for this assay | |
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