Recombinant Human Active Raf-1 (Y340E Y341E, 306-end), CF

R&D Systems | Catalog # 4540-KS

R&D Systems
100% Guarantee

Key Product Details

  • R&D Systems Sf 9 (baculovirus)-derived Recombinant Human Active Raf-1 (Y340E Y341E, 306-end) (4540-KS)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

Sf 9 (baculovirus)

Accession Number

NM_002880

Applications

Bioactivity
View all Raf-1 Proteins and Enzymes »

Product Specifications

Source

Spodoptera frugiperda, Sf 9 (baculovirus)-derived human Raf-1 protein
aa 306-648

Purity

>85%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

N-terminal Sequence Analysis

Using an N-terminal GST tag

SDS-PAGE

63 kDa

Activity

The specific activity of Raf-1 is typically 5100-6900 nmol/min/mg using a myelin basic protein (MBP) substrate (see Activity Assay Protocol).

Scientific Data Images for Recombinant Human Active Raf-1 (Y340E Y341E, 306-end), CF

Recombinant Human Active Raf-1 (Y340E Y341E, 306-end) SDS-PAGE

Recombinant Human Active Raf-1 (Y340E Y341E, 306-end) SDS-PAGE.

The approximate molecular weight is 63 kDa and the purity is > 85%.

Formulation, Preparation, and Storage

4540-KS
Formulation Supplied in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.25 mM DTT, 10 mM Glutathione, 0.1 mM EDTA, 0.1 mM PMSF, and 25% Glycerol.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage This product is stable at ≤ ‑70 °C for up to 1 year from the date of receipt. For optimal storage, aliquot into smaller quantities after centrifugation and store at recommended temperature. Avoid repeated freeze-thaw cycles.

Background: Raf-1

Raf-1 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 Raf-1 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).

References

  1. Rapp, U. et al. (1983) Proc. Natl. Acad. Sci. USA 80:4218.
  2. Li, P. et al. (1991) Cell 64:479.

Long Name

v-raf-1Murine Leukemia Viral Oncogene Homolog 1

Alternate Names

c-Raf-1, Raf1, v-Raf-1

Entrez Gene IDs

5894 (Human); 110157 (Mouse); 24703 (Rat)

Gene Symbol

RAF1

UniProt

NM_002880 (Human)

Additional Raf-1 Products

Product Documents for Recombinant Human Active Raf-1 (Y340E Y341E, 306-end), CF

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Note: Certificate of Analysis not available for kit components.

Product Specific Notices for Recombinant Human Active Raf-1 (Y340E Y341E, 306-end), CF

For research use only

Citations for Recombinant Human Active Raf-1 (Y340E Y341E, 306-end), CF

Customer Reviews for Recombinant Human Active Raf-1 (Y340E Y341E, 306-end), CF

There are currently no reviews for this product. Be the first to review Recombinant Human Active Raf-1 (Y340E Y341E, 306-end), CF and earn rewards!

Have you used Recombinant Human Active Raf-1 (Y340E Y341E, 306-end), CF?

Submit a review and receive an Amazon gift card!

$25/€18/£15/$25CAN/¥2500 Yen for a review with an image

$10/€7/£6/$10CAN/¥1110 Yen for a review without an image

Submit a review
Amazon Gift Card

Protocols

View specific protocols for Recombinant Human Active Raf-1 (Y340E Y341E, 306-end), CF (4540-KS):

Materials
  • Active Kinase - Active Raf-1(EE) (0.1 μg/μL) diluted with Kinase Dilution Buffer III and assayed as outlined in Figure 2. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
  • Kinase Assay Buffer I - 25 mM MOPS, pH 7.2, 12.5 mM beta -glycerolphosphate, 25 mM MgCl2, 5 mM EGTA, 2 mM EDTA. Add 0.25 mM DTT to the Kinase Assay Buffer prior to use.
  • Kinase Dilution Buffer III - Kinase Assay Buffer I diluted at a 1:4 ratio (5-fold dilution) with 50 ng/μL BSA solution.
  • 10 mM ATP Stock Solution - Prepare the ATP Stock Solution by dissolving 55 mg of ATP in 10 mL of Kinase Assay Buffer I. Store 200 μL aliquots at ≤ -20 °C.
  • [33P]-ATP Assay Cocktail - Prepare 250 μM [33P]-ATP Assay Cocktail in a designated radioactive work area by combining 150 μL of 10 mM ATP Stock Solution, 100 μL of [33P]-ATP (1 mCi/100 μL), and 5.75 mL of Kinase Assay Buffer I. Store 1.0 mL aliquots at ≤ -20 °C.
  • Substrate - Inactive MEK1 and ERK1 were activated in a coupled reaction. Myelin Basic Protein (MBP) substrate diluted in distilled or deionized water to a final concentration of 1.0 mg/mL was subsequently used as a substrate for the activated ERK1.
  1. Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
  2. Thaw the Active Raf-1 (EE), Kinase Assay Buffer I, inactive ERK1, and inactive MEK1 on ice.
  3. In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 μL:
    a. Diluted Active Raf-1(EE): 10 μL
    b. Inactive ERK1 (0.2 μg/mL): 1.0 μL
    c. Inactive MEK1 (0.2 μg/mL): 1.0 μL
    d. Kinase Dilution Buffer: 8.0 μL
  4. Start the reaction with the addition of 5.0 μL ATP (250 μM) and incubate in a water bath at 30 °C for 15 minutes.
  5. After the 15 minute incubation, remove 5.0 μL and add it to the following reaction components on ice, bringing the initial reaction volume up to 20 μL:
    a. Reaction Mixture: 5.0 μL
    b. MBP Substrate (1.0 mg/mL; on ice): 5.0 μL
    c. Distilled or deionized water (on ice): 10 μL
  6. Set up the blank control as outlined in Step 5, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled or deionized water.
  7. Initiate the reaction with the addition of 5.0 μL [33P]-ATP Assay Cocktail, bringing the final volume up to 25 μL. Incubate the mixture in a water bath at 30 °C for 15 minutes.
  8. 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.
  9. Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (add 10 mL of phosphoric acid to 990 mL of distilled or deionized water) with constant gentle stirring. It is recommended that the strips be washed a total of three times for approximately 10 minutes each.
  10. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
  11. Determine the corrected cpm by removing the blank control value (see Step 6) for each sample and calculate the kinase specific activity as outlined below.


    Calculation of [33P]-ATP Specific Activity (SA) (cpm/pmol)
    Specific Activity (SA) = cpm for 5.0 μL [33P]-ATP/pmol of ATP (in 5.0 μL of a 250 μM ATP stock solution; i.e. 1250 pmol)

    Calculation of Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
    Corrected cpm from reaction / [(SA of 33P-ATP in cpm/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)] x [(Reaction volume) / (Spot Volume)]

FAQs

No product specific FAQs exist for this product.

View all FAQs for Proteins and Enzymes