Recombinant Human BRAF/B-Raf Protein (GST Tag), Active, CYT-P15056
Product Introduction
BRAF is a member of the Raf/mil family of serine/threonine protein kinases and functions as a key component of the RAS-RAF-MEK-ERK (MAP kinase/ERK) signaling pathway, which regulates critical cellular processes including proliferation, differentiation, migration, and apoptosis. Oncogenic mutations in BRAF, most notably the V600E substitution, result in constitutive kinase activation and are driving events in numerous human cancers, including malignant melanoma, colorectal cancer, non-small cell lung carcinoma, thyroid carcinoma, and non-Hodgkin lymphoma. This recombinant human BRAF protein, encompassing the C-terminal kinase domain (amino acids 416-766), is expressed in baculovirus-infected Sf9 insect cells with an N-terminal GST tag. The purified protein is provided as a constitutively active kinase for in vitro kinase activity assays, inhibitor screening, and drug discovery applications targeting oncogenic BRAF signaling. The insect cell expression system ensures proper eukaryotic post-translational modifications essential for kinase activity and pharmacological relevance.
Product Features
1.Constitutively Active Kinase Domain: Comprises the catalytic kinase domain (amino acids 416-766) with robust enzymatic activity suitable for in vitro kinase assays and inhibitor screening.
2.N-Terminal GST Tag: Enables facile detection and purification using glutathione-based methods, with the option for tag cleavage if desired for downstream applications.
3.Baculovirus-Insect Cell Expression: Produced in Sf9 insect cells, providing eukaryotic folding and post-translational modifications critical for physiological kinase activity.
4.Validated Kinase Activity: Active kinase suitable for screening small-molecule BRAF inhibitors, studying substrate phosphorylation, and biochemical pathway reconstitution.
5.Lot-Specific Quality Data: Each batch is accompanied by a detailed technical data sheet providing lot-specific purity and specific activity information.
Specifications
Size: Available in 10 µg and 50 µg
Catalog Number: CYT-P15056
Protein Construct: Recombinant human BRAF amino acids 416-766 with N-terminal GST tag
Expression Host: Baculovirus-infected Sf9 insect cells
Species: Human
Molecular Weight: ~63 kDa (GST-tagged kinase domain)
Buffer: 50 mM Tris-HCl, pH 7.5, 50-300 mM NaCl, 10 mM glutathione, 0.1 mM EDTA, 0.25-1.0 mM DTT, 0-0.1 mM PMSF, 10-25% glycerol
Purity: For lot-specific information, see the accompanying technical data sheet.
Biological Activity: For lot-specific information, see the accompanying technical data sheet.
Storage and Stability
Storage Conditions: Store at -70¡ãC. Upon receipt, briefly centrifuge the vial to collect contents at the bottom. Aliquot into single-use volumes and store at -70¡ãC. Avoid repeated handling and multiple freeze/thaw cycles.
Shipping Conditions: Shipped on dry ice to preserve enzymatic activity. Ambient temperature shipment is not recommended and may compromise protein activity.
Shelf Life: The product is stable for 12 months from the date of manufacture when stored as directed at -70¡ãC.
Protocol (For Reference Only)
Important: This protein is an active kinase; handle at 4¡ãC or on ice. Minimize freeze-thaw cycles by aliquoting upon initial reconstitution. Kinase activity is lot-specific; consult the batch-specific technical data sheet for activity information.
1.Thawing and Handling: Thaw the protein aliquot on ice. Briefly centrifuge to collect contents at the bottom of the vial before opening. Do not vortex; gently pipette to mix.
2.Kinase Activity Assay Buffer: Prepare kinase assay buffer typically containing 25-50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, and 0.1 mg/mL BSA. Include ATP at concentrations appropriate for the specific assay format (typically 10-100 µM for screening applications).
3.Kinase Activity Measurement: Dilute BRAF protein to the desired concentration in assay buffer (typically 10-100 ng per reaction for radiometric or fluorescence-based kinase assays). Add substrate (e.g., inactive MEK1 or generic peptide substrate) and ATP to initiate the reaction. Incubate at 30¡ãC for 15-60 minutes. Stop the reaction by adding EDTA or spotting onto phosphocellulose paper, and quantify phosphate incorporation by appropriate detection method.
4.Inhibitor Screening: For compound screening, pre-incubate BRAF kinase with test compounds or reference inhibitors (e.g., vemurafenib, dabrafenib) in assay buffer for 10-15 minutes at room temperature before initiating the reaction with ATP and substrate. Calculate IC₅₀ values from dose-response curves.
5.GST Tag Detection and Purification: The N-terminal GST tag enables detection by anti-GST Western blot and purification by glutathione-Sepharose affinity chromatography. For applications where the GST tag may interfere, consult the technical data sheet for tag cleavage options.
Precautions
1.This protein is an active kinase; minimize time at room temperature and always thaw on ice.
2.Aliquot the protein into single-use volumes after initial thawing to avoid repeated freeze-thaw cycles, which significantly reduce kinase activity.
3.The exact buffer composition, purity, and specific activity are lot-specific; always consult the batch-specific technical data sheet and certificate of analysis for detailed information before use.
4.Do not use if the dry ice has completely sublimated upon arrival; contact technical support for guidance.
5.For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: What region of BRAF does this protein encompass?
A1: This recombinant protein comprises the C-terminal kinase domain (amino acids 416-766) of human BRAF with an N-terminal GST tag. The N-terminal regulatory domain is not included, and the kinase domain is constitutively active without the need for RAS activation.
Q2: Is this protein the wild-type or V600E mutant?
A2: This protein represents the wild-type kinase domain sequence (amino acids 416-766). For the V600E mutant or other oncogenic variants, please inquire separately. Contact the supplier for information on available mutant constructs.
Q3: How should I determine the specific activity of my lot?
A3: Specific activity and purity data are lot-specific and provided in the accompanying technical data sheet and certificate of analysis (COA). Refer to the lot number on your vial to access the corresponding documentation.
Q4: Can the GST tag be removed?
A4: The GST tag is fused to the N-terminus of the kinase domain. A protease cleavage site is typically engineered between the GST tag and the BRAF sequence. Consult the lot-specific technical data sheet for information on tag cleavage feasibility and recommended proteases.
Disclaimer
1.For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2.Due to the variable nature of biological and biochemical research, optimization of assay conditions is strongly recommended for specific experimental systems.
3.This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4.Wear appropriate protective clothing and gloves when handling this product.
Ordering Information
Catalog Number: CYT-P15056
Product Name: Recombinant Human BRAF/B-Raf Protein (GST Tag), Active
Size: 10 µg / 50 µg
Price:
10 µg: CNY ¥4550.00 / USD $455.00 / EUR €546.00 / JPY ¥81900.00
50 µg: CNY ¥13600.00 / USD $1360.00 / EUR €1632.00 / JPY ¥244800.00
