Kinase Assay Kit, BioAssay(TM)

Kinases, also known as phosphotransferases, constitute a large family of enzymes that transfer phosphate groups from the high-energy donor molecule ATP, to their specific substrates. Kinases are known to regulate the majority of cellular processes. The largest group of this family is the protein kinases. So far, 518 different kinases have been identified in humans and up to 30% of human proteins are modified by these kinases. The enormous diversity and their key role in cellular signaling make them ideal targets for drug developments.\nKinase Assay Kit provides a simple and rapid method for assaying kinase activity and high-throughput screening for kinase inhibitors. This homogeneous microplate-based assay involves incubating the kinase with a single working reagent, in which ADP is enzymatically converted to ATP and pyruvate, which is quantified using a fluorimetric (530nm/590nm) assay method.\n\nKey Features:\nSafe. Non-radioactive assay.\nSensitive and accurate. As low as 0.01 U/L kinase can be quantified.\nHomogeneous and convenient. "Mix-incubate-measure" type assay. The whole assay involves adding a single working reagent and incubation for 10 min at room temperature.\nRobust and amenable to HTS: Assay can tolerate up to 300uM ATP and 10% dimethylsulfoxide (DMSO). ZпїЅ factors of > 0.6 are routinely observed in 96/384-well plates. Can be readily automated on HTS liquid handling systems for tens of thousands of assays per day.\n\nApplications:\nKinase activity assays and HTS for kinase inhibitors.\nADP determination in cells and other biological samples.\n\nKit Contents:\nReagent A: 10ml Reagent B: 10ml\nAssay Buffer: 25ml Standard: 100ul 3mM ADP\nStorage conditions: store all reagents at -20 degrees C. This product is shipped on dry ice. Shelf life: 6 months after receipt.\nPrecautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.\n\nAssay Procedure:\nThis kit is sufficient for 400 assays in 384-well plate (200 assays in 96-well plate). Use black flat-bottom plates. Prior to assay, bring all reagents to room temperature. Assays in duplicate wells are recommended. Interference: thiols (b-mercaptoethanol, dithioerythritol etc) at > 10uM interfere with this assay and should be avoided. \nKinase Activity Assay in 384-well Plate\n1. Kinase Reaction. Users should provide their own enzyme, ultra-pure ATP and substrate. Set up 20ul reaction mixture containing the kinase, ATP and substrate in the provided Assay Buffer (pH 7.0) or any suitable kinase buffer. Set up a Blank Control that contains ATP and substrate but no enzyme. Incubate at desired temperature for desired period of time e.g. 30 min.\n2. Standards. Prepare 900ul 10uM ADP Premix by mixing 3ul 3mM Standard and 897ul distilled water. Dilute standard as follows. Transfer 20ul standards into separate wells of the plate.\nNo Premix+H2O Vol (ul) ADP (uM)\n1 100ul+0ul 100 10\n2 60ul+40ul 100 6\n3 30ul+70ul 100 3\n4 0ul+100ul 100 0\n3. ADP Detection. Prepare enough Working Reagent for each well by mixing 25ul Reagent A and 25ul Reagent B. Add 40ul Working Reagent to each assay well. Tap plate to mix. Incubate at room temperature for 10 min.\n4. Read fluorescence intensity at lexc=530nm and lem=590nm. Calculate kinase activity,\nKinase Activity =\nDFSAMPLE\nSlope пїЅ t\nx (U/L)\n20ul\nVol (ul)\nwhere DFSAMPLE=(fluorescence intensity of sample wellпїЅfluorescence of the blank well), slope is the slope of the ADP standard curve. t is the kinase reaction time (e.g. 30 min). Vol is the volume (ul) of kinase added to the 20ul reaction.\nNote: typical kinase assays use 100 to 200uM ATP. If DFSAMPLE > (F10uM ADPпїЅFH2O), dilute enzyme in assay buffer. Repeat the assay, multiply the results by the dilution factor n. This will ensure that the initial rate is measured at < 10% ATP conversion.\nHigh-throughput Screen for Kinase Inhibitors\nFor inhibitor screens, test compounds are usually pre-incubated with kinase for 10 to 30 min, prior to adding ATP/substrate to initiate kinase reaction. After a 30-min kinase reaction, the produced ADP is quantified using the Fluorimetric Assay:. The fluorescence intensity will be decreased in the presence of an inhibitor.\n1. Controls and compounds. Use a known kinase inhibitor (e.g. staurosporine) as a positive control. Alternatively, пїЅno enzymeпїЅ wells can serve as a positive control. Use the same volume of the compound solvent (e.g. DMSO) as an inhibitor negative control. Example: transfer 5ul test compound, control inhibitor and negative control (e.g. DMSO) to appropriate wells, add 10ul enzyme solution to all assay wells. Apply an пїЅin-wellпїЅ mixing step. Incubate for 10 to 30 min.\n2. Kinase Reaction. Add 5ul mixture containing ATP and the kinase substrate to each assay well. Mix well. Incubate for desired period of time (e.g. 30 min).\n3. ADP Detection. Prepare enough Working Reagent by mixing 25ul Reagent A and 25ul Reagent B. Add 40ul Working Reagent to each well and mix well. Incubate for 10 min.\n4. Read fluorescence intensity at lexc=530nm and lem=590nm.

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SPECIFICATIONS

Catalog Number

K1774-10

Size

1Kit

References

1. Manning G. et al. (2002). The protein kinase complement of the human genome. Science 298: 1912-1934.\n2. Grant SK. (2009). Therapeutic protein kinase inhibitors. Cell Mol Life Sci. 66(7): 1163-1177.