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Application Notes
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Note: these are suggested working dilutions and it is recommended that the researcher perform a serial dilution of Active PDGFRbeta for optimal results). [ 32 P]-ATP Assay Cocktail Prepare 250μM [ 32 P]-ATP Assay Cocktail in a designated radioactive working area by adding the following components: 150μl of 10mM ATP Stock Solution, 100μl [ 32P ]- ATP (1mCi/100μl), 5.75ml of Kinase Assay Buffer. Store 1ml aliquots at –20 °C. Kinase Dilution Buffer, pH 7.2 Kinase Assay Buffer II diluted at a 1:4 ratio (5X dilution) with 50 ng/μl BSA solution. 10mM ATP Stock Solution Prepare ATP stock solution by dissolving 55mg of ATP in 10ml of Kinase Assay Buffer. Store 200μl aliquots at –20 °C. Kinase Assay Buffer II, pH 7.2 Buffer components: 25mM MOPS, 12.5mM beta-glycerol-phosphate, 20mM MgC1 2 , 25mM MnC1 2 , 5mM EGTA, 2mM EDTA. Add 0.25mM DTT to Kinase Assay Buffer prior to use. Substrate Poly (Glu:Tyr, 4:1) synthetic peptide substrate diluted in distilled H 2 O to a final concentration of 1 mg/ml. Assay Protocol Step 1. Thaw [ 32 P]-ATP Assay Cocktail in shielded container in a designated radioactive working area. Step 2. Thaw the Active PDGFRbeta, Kinase Assay Buffer, Substrate and Enzyme Dilution Buffer on ice. Step 3. In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20μl: Component 1. 10μl of diluted Active PDGFRbeta. Component 2. 10μl of 1 mg/ml stock solution of substrate Step 4. Set up the blank control as outlined in step 3, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled H 2 O. 0 60,000 120,000 180,000 240,000 300,000 0 200 400 600 800 Protein (ng) Activity (CPM) FOR IN VITRO RESEARCH PURPOSES ONLY. NOT INTENDED FOR USE IN HUMAN OR ANIMALS. Step 5. Initiate the reaction by the addition of 5μl [ 32 P]-ATP Assay Cocktail bringing the final volume up to 25μl and incubate the mixture in a water bath at 30 °C for 15 minutes. Step 6. After the 15 minute incubation period, terminate the reaction by spotting 20μl of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper. Step 7. Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (dilute 10ml of phosphoric acid and make a 1L solution with distilled H 2 O) with constant gentle stirring. It is recommended that the strips be washed a total of 3 intervals for approximately 10 minutes each. Step 8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter. Step 9. Determine the corrected cpm by removing the blank control value (see Step 4) for each sample and calculate the kinase specific activity as outlined below. Calculation of [P 32 ]-ATP Specific Activity (SA) (cpm/pmol) Specific activity (SA) = cpm for 5μl [ 32 P]-ATP / pmoles of ATP (in 5μl of a 250μM ATP stock solution, i.e., 1250 pmoles) Kinase Specific Activity (SA) (pmol/min/μg or nmol/min/mg) Corrected cpm from reaction / [(SA of 32 P-ATP in cpm/pmol)*(Reaction time in min)*(Enzyme amount in μg or mg)]*[(Reaction Volume) / (Spot Volume)]
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Buffer
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Recombinant protein stored in 50mM Tris-HCl, pH 7.5, 150mM NaCl, 0.25mM DTT, 0.1mM EGTA, 0.1mM EDTA, 0.1mM PMSF, 25% glycerol
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