| Assay Method Information | |
| | Enzyme Assay: Pan SIK MS IC50 Assay |
| Description: | Salt Induced Kinase (SIK) activity is determined by measuring the effect of a test agent on the activity of the appropriate SIK enzyme to phosphorylate its corresponding substrate. The substrate peptides AQT0868, AQT0252, and AQT0508 were engineered by AssayQuant to be specific for their corresponding enzymes SIK 1, SIK 2, and SIK 3 respectively. The phosphorylated substrate product resulting from the kinase reaction is detected by LCMS/MS, and the area under the peak curve correlates with kinase activity. Before initiating the assay, the assay ready plates (ARP) containing 0.15 μL compound in dose response format (10 μM to 9.5 pM by 4-fold dilution) are thawed for 20-30 minutes at room temperature. The plates are spun at 1000 rpm for 30 seconds to ensure compounds are at the bottom of the well before removing foil covers. 0.15 μL HPE (assay std) and ZPE (DMSO) are added to the ARP using an HP D300 dispenser. Plates are spun again at 1000 rpm for 30 seconds. 20 μL of 1.25× enzyme (one plate each for SIK 1, SIK 2, and SIK 3) with 1 mM ATP in reaction buffer (Reagent Grade Water, 50 mM Hepes pH 7.5, 0.5 mM EGTA, 10 mM MgCl2, 0.01% Brij-35, 1% Glycerol, 0.01% BSA, 1 mM TCEP) are added with a Multidrop Combi and designated small volume cassette. Plates are spun at 1000 rpm for 30 seconds. Plates are then sealed and incubated at RT for 10 minutes. Following incubation, 5 μL of 5× peptide reagent is added with a Multidrop Combi and designated small volume cassette to initiate the kinase reaction. Plates are spun at 1000 rpm for 30 seconds. Plates are then sealed and incubated at RT for 90 minutes. The assay is stopped by the addition of 5 μl of 120 mM EDTA added with a Multidrop Combi and designated small volume cassette (20 mM final concentration). Plates are spun at 1000 rpm for 30 seconds. 10 μl of the final reaction mix from each of the three individual enzyme plates are transferred by a Platemate Plus to the same corresponding 96 well deepwell plate containing 70 μl Water/Methanol (85/15%) for multiplex MS detection (100 μl total volume). Using Activity Base (ABase), an idbs data analysis software tool, the reported area ratio unit data is associated to compound, batch and dose information, the data is evaluated for quality, and the percent inhibition of each well is calculated. The data from each Max effect/HPE and Min effect/ZPE control wells are assessed, and outliers are excluded from all further calculations. The mean and standard deviation of the HPE and ZPE are then calculated for each plate, along with the Z Prime. The compound data is converted into % effect, using the average ZPE and HPE controls as 0% and 100% activity, respectively. |
| Affinity data for this assay | |
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