产品名称：ASK1, GST-FUSION, HU.,REC.,S. FRUGIPERDA
Application Notes: Activity Assay
• Kinase Assay Buffer: 25 mM MOPS, 25 mM MgCl2, 12.5 mM β-glycerophosphate, 5 mM EGTA, 2 mM EDTA, pH 7.2; add 0.25 mM DTT just prior to use
• Kinase Dilution Buffer: Kinase Assay Buffer diluted 1:5 with 50 ng/µl BSA
• ASK1: Prepare serial dilutions using Kinase Dilution Buffer as outlined in the sample data below or as deemed necessary for individual experimental conditions
• 10 mM ATP Stock Solution: Dissolve 55 mg ATP in 10 ml Kinase Assay Buffer; dispense into 200 µl aliquots and freeze (-20°C)
• 250 µM [32P]-ATP Assay Cocktail: 150 µl 10 mM ATP stock solution, 100 µl [32P]-ATP (1 mCi/100 µl), 5.75 ml Kinase Assay Buffer; dispense into 1 ml aliquots and freeze (-20°C)
• Substrate: Dissolve myelin basic protein (MBP) in dH2O to a final concentration of 1 mg/ml
Activity Assay Protocol
1. Thaw an aliquot of [32P]-ATP Assay Cocktail in a shielded container in a designated radioactive working area.
2. Thaw the ASK1, Kinase Assay Buffer, Substrate, and Enzyme Dilution Buffer on ice.
3. In a pre-cooled tube, add the following components:
• 10 µl diluted ASK1
• 10 µl 1 mg/ml Substrate stock
4. Prepare a Blank as outlined in step 3, replacing the 10 µl Substrate with an equal volume of water.
5. Initiate the reaction by adding 5 µl [32P]-ATP Assay Cocktail (final volume is 25 µl) and incubating in a water bath at 30°C for 15 min.
6. Terminate the reaction by spotting 20 µl of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.
7. Air-dry the pre-cut strips and sequenentially wash in 1% phosphoric acid with constant, gentle stirring for 10 min. Repeat for a total of 3 washes.
8. Count the radioactivity in presence of scintillation fluid.
9. Determine the corrected cpm by substracting the value of the Blank from each sample and calculate as follows:
Presentation: Liquid. In 150 mM NaCl, 50 mM Tris-HCl, 0.25 mM DTT, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol, pH 7.5.
Unit Definition: Kinase activity is measured as the molar amount of phosphate incorporated into MBP at 30°C using a final concentration of 50 µM ATP.