After the 15 minute incubation, terminate the reaction by spotting 20 μL of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.Incubate the mixture in a water bath at 30 ☌ for 15 minutes. Initiate the reaction with the addition of 5 μL -ATP Assay Cocktail, bringing the final volume up to 25 μL.Replace the substrate with an equal volume of distilled or deionized water. Set up the blank control as outlined in step 3, excluding the addition of the substrate.In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 μL.
Thaw the Active PKA C alpha, Kinase Assay Buffer, Substrate, and Kinase Dilution Buffer on ice.Thaw the -ATP Assay Cocktail in a shielded container in a designated radioactive work area.Substrate - CREBtide synthetic peptide substrate (KRREILSRRPSYR) diluted in distilled or deionized water to a final concentration of 1 mg/mL.-ATP Assay Cocktail - Prepare 250 μM -ATP Assay Cocktail in a designated radioactive work area by combining 150 μL of 10 mM ATP Stock Solution, 100 μL of -ATP (1 mCi/100 μL), and 5.75 mL of Kinase Assay Buffer.10 mM ATP Stock Solution - Prepare the ATP Stock Solution by dissolving 55 mg of ATP in 10 mL of Kinase Assay Buffer.Kinase Dilution Buffer, pH 7.2 - Kinase Assay Buffer diluted 5-fold with a 50 ng/μL BSA and 5% glycerol solution.Add 0.25 mM DTT to the Kinase Assay Buffer prior to use.
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