Recombinant Human B-Raf kinase domain His-tag Protein, CF

R&D Systems | Catalog # 11831-BR

R&D Systems

Key Product Details

Source

Sf 21 (baculovirus)

Applications

Enzyme Activity
View all B-Raf Proteins and Enzymes »

Product Specifications

Source

Spodoptera frugiperda, Sf 21 (baculovirus)-derived human B-Raf protein
Met 6-His tag Sumo-tag
(mutated, uncleavable)		 3c Protease site Human B-Raf
(Leu416-His766)
Accession # P15056.4
N-terminus C-terminus

Purity

>85%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<0.10 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Protein identity confirmed by mass spectrometry

Predicted Molecular Mass

53 kDa

SDS-PAGE

49-58 & 27-33 kDa, under reducing conditions

Activity

Measured by its ability to transfer phosphate from adenosine triphosphate (ATP) to a synthetic peptide substrate.
The specific activity is >20 pmol/min/μg, as measured under the described conditions.

Scientific Data Images for Recombinant Human B-Raf kinase domain His-tag Protein, CF

Recombinant Human B‑Raf kinase domain His-tag Enzyme Activity.

Recombinant Human B‑Raf kinase domain His-tag (Catalog # 11831-BR) is measured by its ability to transfer phosphate from adenosine triphosphate (ATP) to a synthetic peptide substrate.

Formulation, Preparation, and Storage

11831-BR
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, DTT and Glycerol.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: B-Raf

Serine/Threonine-protein kinase B-Raf (B-Raf), also referred to as proto-oncogene B-Raf and v-Raf murine sarcoma viral oncogene homolog B is a broadly expressed cytoplasmic kinase from the TKL (Tyrosine Kinase-Like) group of Serine/Threonine protein kinase family that functions as a critical gatekeeper in the MAP kinase/ERKs signaling pathway that controls cell division, proliferation and survival (1,2). B-Raf is one of three highly conserved RAF kinase isoforms that are composed of three conserved domains characteristic of the subfamily (3). Conserved region 1 (CR1) contains a Ras-GTP-binding self-regulatory domain that autoinhibits B-Raf's kinase domain thus regulating B-Raf signaling (3,4). Conserved region 2 (CR2) is a serine-rich hinge region, and conserved region 3 (CR3), is a conserved bi-lobal catalytic protein kinase domain where the smaller lobe contains the P-loop responsible for ATP binding and the larger lobe contains the activation loop that binds substrate proteins with the active site located in the cleft between the two lobes (5). B-Raf must bind Ras-GTP which causes a conformational change to its active dimeric conformation and phosphorylation (6,7). Although similar in structure, B-Raf is the Raf family member that has been shown to function as the main and most potent activator of the MAPK cascade via targeted MEK phosphorylation (3,8). Inherited mutations in the BRAF gene can cause Cardiofaciocutaneous, Noonan, and Leopard syndromes (9). More than 30 mutations of the BRAF gene, including the V600E mutation within the activation segment that causes constitutive signaling, have been associated with diverse human cancer types including colorectal cancer, melanoma, papillary thyroid carcinoma, non-small-cell lung carcinoma, glioblastoma (10-14), as well as inflammatory diseases such as Erdheim–Chester disease (1,2,15). As BRAF is a well-understood target (16) and inhibition via approved drugs targeting B-Raf specifically and in combination have shown improved survival and response rate for several mutation-related cancers including solid tumors (1,17,18,19), there is significant ongoing research in development of additional pharmacological inhibitors for B-Raf kinase.   

References

  1. Poulikakos, P. et al. (2022) Clin. Cancer Res. 28:4618. 
  2. Mechahougui, H. et al. (2025) Int. J. Sci. 26:3757. 
  3. Daum, G. et al. (1994) Trends Biochem. Sci. 19:474. 
  4. Cutler Jr, R.E. et al. (1998) Proc. Natl. Acad. Sci. USA 95:9214. 
  5. Hanks, S.K. and Hunter, T. (1995) FASEB J. 9:576. 
  6. Morrison, D.K. and Cutler, R.E. (1997) Curr. Opin. Cell Biol. 9:174. 
  7. Lavoie, H. and Therrien, M. (2015) Nat. Rev. Mol. Cell Biol. 16:281. 
  8. Baccarini, M. (2005) FEBS Lett. 579:3271. 
  9. Sarkozy, A. et al. (2009) Hum. Mutat. 30:695. 
  10. Davies, H. et al. (2002) Nature 417:949. 
  11. Marchetti, A. et al. (2011) J. Clin. Oncol. 29:3574. 
  12. Choi, S. et al. (2016) Int. J. Radiat. Oncol. Biol. Phys. 96:E356. 
  13. Chu, J.E. et al. (2020) Clin. Cancer Res. 26:4599. 
  14. Lim-Fat, M.J. et al. (2021) J. Neurooncol. 152:515.
  15. Haroche, J. et al. (2017) Lancet Oncol. 18:e113. 
  16. King, A.J. et al. (2006) Cancer Res. 66:11100. 
  17. Chapman, P.B. et al. (2011) N. Engl. J. Med. 364:2507. 
  18. Barbato, M.I. et al. (2024) Clin. Cancer Res. 30:263. 
  19. Hanrahan, A.J. et al. (2024) Nat. Rev. Clin. Oncol. 21:224.

Long Name

v-Raf Murine Sarcoma Viral Oncogene Homolog B1

Alternate Names

BRaf, BRAF1, RAFB1

Entrez Gene IDs

673 (Human); 114486 (Rat)

Gene Symbol

BRAF

Additional B-Raf Products

Product Documents for Recombinant Human B-Raf kinase domain His-tag Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

Product Specific Notices for Recombinant Human B-Raf kinase domain His-tag Protein, CF

For research use only

Related Research Areas

Cancer Biomarkers
Intracellular Kinases

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Protocols

View specific protocols for Recombinant Human B-Raf kinase domain His-tag Protein, CF (11831-BR):

Materials
  • Assay Buffer: 50 mM Tris, 20 mM MgCl2, 5 mM MnCl2, 0.1 mg/mL BSA, pH 7.5
  • Recombinant Human B-Raf kinase domain His-tag Protein (rhB-Raf) (Catalog # 11831-BR)
  • Poly (4:1 Glu, Tyr), 1 mg/mL stock in 25 mM Tris, pH 7.5
  • Adenosine triphosphate (ATP), 10 mM stock in deionized water
  • ADP-GloTM Kinase Assay Kit (Promega)
  • White 96-well Plate
  • Plate Reader with Luminescence Read Capability
  1. Dilute rhB-Raf to 20 µg/mL in Assay Buffer.
  2. Prepare Substrate Mixture containing 200 µM ATP and 25 µg/mL Poly (4:1 Glu, Tyr) in Assay Buffer.
  3. Combine equal volumes of 20 µg/mL rhB-Raf and Substrate Mixture. Create a Substrate Control containing equal volumes of Assay Buffer and Substrate Mixture.
  4. Incubate at room temperature for 40 minutes in the dark.
  5. After incubation, transfer 10 µL of each reaction to wells of a white plate.
  6. Terminate the reaction and deplete the remaining ATP by adding 10 µL of ADP-GloTM Reagent (supplied in kit) to all wells.
  7. Incubate at room temperature for 40 minutes in the dark.
  8. Add 20 µL Kinase Detection Reagent (supplied in kit) to all wells.
  9. Incubate at room temperature for 30 minutes in the dark.
  10. Read plate in Luminescence endpoint mode.
  11. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

Adjusted Luminescence* (RLU) x Conversion Factor** (pmol/RLU)
Incubation time (min) x amount of enzyme (µg)

    

*Adjusted for Substrate Control
**Derived from ADP-GloTM Kinase Assay Kit protocol (Promega)
Per Reaction:
  • rhB-Raf: 10 µg/mL 
  • ATP: 100 µM
  • Poly (4:1 Glu, Tyr): 12.5 µg/mL




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