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

Details for Product No. ABIN457520, Supplier: Log in to see
Protein Name
  • CRAF
  • NS5
  • Raf-1
  • c-Raf
  • c-raf
  • craf
  • raf1
  • RAF1
  • 6430402F14Rik
  • AA990557
  • BB129353
  • Craf1
  • D830050J10Rik
  • v-Raf
  • raf
  • cRaf
  • Raf-1 proto-oncogene, serine/threonine kinase
  • Raf-1 proto-oncogene, serine/threonine kinase a
  • RAF proto-oncogene serine/threonine-protein kinase pseudogene
  • v-raf-leukemia viral oncogene 1
  • Raf-1 proto-oncogene, serine/threonine kinase S homeolog
  • RAF1
  • Raf1
  • raf1a
  • raf1
  • LOC100341921
  • raf1.S
Origin
Human
10
5
1
1
Source
Baculovirus infected Insect Cells
5
3
2
1
1
1
1
Protein Type
Recombinant
Biological Activity
Active
Application
Functional Studies (Func), SDS-PAGE (SDS), Western Blotting (WB)
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Purpose >85% Pure active Recombinant RAF1.
Characteristics Protein Source: Baculovirus (Sf9 insect cells)
Purity SDS-PAGE: ≥85 %
Background RAF1 is a MAP kinase kinase kinase (MAP3K), which functions downstream of the Ras family of membrane associated GTPases to which it binds directly (1). The activated RAF1 can phosphorylate and activate the dual specificity protein kinases MEK1 and MEK2, which in turn phosphorylate to activate the serine/threonine specific protein kinases ERK1 and ERK2. Activated ERKs are pleiotropic effectors of cell physiology and play an important role in the control of gene expression involved in the cell division cycle, apoptosis, cell differentiation and cell migration (2).
Alternate Names: RAF, RAF proto-oncogene serine/threonine-protein kinase , Proto-oncogene c-RAF
Molecular Weight 63.0 kDa
Gene ID 5894
UniProt P04049
Research Area Signaling, Kinases/Phosphatases
Pathways MAPK Signaling, RTK Signaling, Fc-epsilon Receptor Signaling Pathway, Neurotrophin Signaling Pathway, cAMP Metabolic Process, Stem Cell Maintenance, Hepatitis C, Autophagy, Signaling of Hepatocyte Growth Factor Receptor, VEGF Signaling
Application Notes Optimal working dilution should be determined by the investigator.
Comment

Activity Specification: The specific activity of RAF1(EE) was determined to be ~6,000 nmol /min/mg in a coupled assay as per activity assay protocol.

Protocol 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: 150 µL of 10 mM ATP Stock Solution, 100 µL [ 32P ]- ATP (0 mCi/100 µL), 5.70 ml of Kinase Assay Buffer I. Store 0 ml 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. 10 mM ATP Stock Solution Prepare ATP stock solution by dissolving 50 mg of ATP in 10 ml of Kinase Assay Buffer I. Store 200 µL aliquots at -20 °C. Kinase Assay Buffer I Buffer components: 20 mM MOPS, pH 7.2, 12.0 mM beta-glycerol-phosphate, 20 mM MgC1 2, 0 mM EGTA, 0 mM EDTA. Add 0.20 mM 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 H2O to a final concentration of 0 mg/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 20 µL: Component 1. 10 µL of diluted Active RAF1(EE) Component 2. 0 µL of Unactive MEK1 (0.2μg/µL) Component 3. 0 µL of Unactive ERK1 (0.2μg/µL) Component
4. 0 µL 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 0 µL and add to the following reaction components bringing the initial reaction volume up to 20 µL on ice: Component 1. 0 µL of reaction mixture Component 2. 10 µL distilled H2O on ice Component 3. 0 µL of MBP substrate on ice(1 mg/mL)
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 H2O.
5. Initiate the reaction by the addition of 0 µL [ 32 P]-ATP Assay Cocktail bringing the final volume up to 20 µL and incubate the mixture in a water bath at 30 °C for 15 minutes.
6. After the 15 minute incubation period, 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 P81 strip and sequentially wash in a 1 % phosphoric acid solution (dilute 10 ml of phosphoric acid and make a 1L solution with distilled H2O) with constant gentle stirring. It is recommended that the strips be washed a total of 3 intervals for approximately 10 minutes each.
8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
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.
Restrictions For Research Use only
Format Liquid
Concentration 0.1 μg/μL
Buffer Recombinant protein stored in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.25 mM DTT, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 25 % glycerol.
Preservative Dithiothreitol (DTT)
Precaution of Use This product contains dithiothreitol (DTT): a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handling Advice Centrifuge the vial prior to opening.
Storage -80 °C
Storage Comment -80°C
Expiry Date 12 months
Supplier Images
Western Blotting (WB) image for V-Raf-1 Murine Leukemia Viral Oncogene Homolog 1 (RAF1) (Active) protein (ABIN457520) V-Raf-1 Murine Leukemia Viral Oncogene Homolog 1 (RAF1) (Active) protein
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