V-Raf-1 Murine Leukemia Viral Oncogene Homolog 1 (RAF1) Protein

Name: V-Raf-1 Murine Leukemia Viral Oncogene Homolog 1 (RAF1)

Synonyme: NS5, CRAF, Raf-1, c-Raf, craf, c-raf, RAF1, MGC165882, raf1, DKFZp469F0314

Biologische Aktivität: Active

Applikation: Enzymaktivitätsmessung (EAA)

Produktnummer: ABIN411933

Anwendungen

Applikationshinweise: Note: these are suggested working dilutions and it is recommended that the researcher perform a serial dilution of Active RAF1(EE) 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 I. Store 1ml aliquots at –20 °C. Kinase Dilution Buffer III Kinase Assay Buffer I diluted at a 1:4 ratio (5X dilution) with 50ng/μl BSA solution. 10mM ATP Stock Solution Prepare ATP stock solution by dissolving 55mg of ATP in 10ml of Kinase Assay Buffer I. Store 200μl aliquots at –20 °C. Kinase Assay Buffer I Buffer components: 25mM MOPS, pH 7.2, 12.5mM beta-glycerol-phosphate, 25mM MgC1 2 , 5mM EGTA, 2mM EDTA. Add 0.25mM DTT to Kinase Assay Buffer prior to use. Substrate Unactive MEK1 and ERK1 were activated in a coupled reaction. Myelin Basic Protein (MBP) diluted in distilled H 2 O to a final concentration of 1mg/ml was subsequently used as a substrate for the activated ERK1. Assay Protocol Step 1. Thaw the Active RAF1(EE), Kinase Assay Buffer, Unactive ERK1 and Unactive MEK1 on ice. 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 RAF1(EE) Component 2. 2μl of Unactive MEK1 (0.2μg/μl) Component 3. 3μl of Unactive ERK1 (0.2μg/μl) Component 4. 5μl of Kinase Dilution Buffer Step 2. Start the reaction by the addition of 5 μl ATP (250μM) and incubate in a water bath at 30 °C for 25 minutes. Step 3. After the 25 minute incubation period, remove 5μl and add to the following reaction components bringing the initial reaction volume up to 20μl on ice: Component 1. 5μl of reaction mixture Component 2. 10μl distilled H 2 O on ice Component 3. 5μl of MBP substrate on ice(1 mg/ml) 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. 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)]

Beschränkungen: Nur für Forschungszwecke einsetzbar