Recombinant Human VEGFR2/KDR kinase domain His Protein, CF

R&D Systems | Catalog # 11740-KD

His-tag
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Key Product Details

Source

Sf 21 (baculovirus)

Accession Number

P35968.2

Applications

Enzyme Activity
View all VEGFR2/KDR/Flk-1 Proteins and Enzymes »

Product Specifications

Source

Spodoptera frugiperda, Sf 21 (baculovirus)-derived human VEGFR2/KDR/Flk-1 protein
Met 6-His tag Sumo-tag
(mutated, uncleavable)		 3c Protease site Human VEGFR2
(Ala792-Val1356)
Accession # P35968.2

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

76 kDa

SDS-PAGE

87-101 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 >225 pmol/min/μg, as measured under the described conditions.

Scientific Data Images for Recombinant Human VEGFR2/KDR kinase domain His Protein, CF

Recombinant Human VEGFR2/KDR/Flk-1 kinase domain His-tag Enzyme Activity.

Recombinant Human VEGFR2/KDR/Flk-1 kinase domain His-tag (Catalog # 11740-KD) is measured by its ability to transfer phosphate from adenosine triphosphate (ATP) to a synthetic peptide substrate.

Recombinant Human VEGFR2/KDR/Flk-1 kinase domain His-tag SDS-PAGE.

2 μg/lane of Recombinant Human VEGFR2/KDR/Flk-1 kinase domain His-tag (Catalog # 11740-KD) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 87-101 kDa, under reducing conditions.

Formulation, Preparation, and Storage

11740-KD
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, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Background: VEGFR2/KDR/Flk-1

Vascular endothelial growth factor receptor 2 (VEGFR2), also known as Fetal liver kinase 1 (FLK-1) and Kinase insert domain receptor (KDR), is one of three ubiquitously expressed, highly conserved tyrosine protein kinase VEGFRs from the platelet-derived growth factor receptor (PDGFR) family (1). VEGFR2, like other receptor members in this family is comprised of an extracellular domain (ECD) with immunoglobulin-like segments that binds its target cytokine VEGF to induce dimerization, a transmembrane domain, and a cytosolic kinase domain (1). Similar to the structure of other tyrosine kinases, the kinase domain contains the conserved catalytic domain with an ATP binding site within the cleft of two lobes at the N-terminal and C-terminal regions as well as the glycine-rich or nucleotide binding loop and activation loop (1,2).  Unique to this family there is also a kinase insert domain (KID) of unknown function (1). Through ligand binding, dimerization, activation via autophosphorylation, and subsequent phosphorylation of targets in multiple signaling pathways, VEGFR2 regulates endothelial cell growth, migration, and survival and plays a critical role in angiogenesis (2-4). Abnormal angiogenesis is a hallmark of tumor progression and metastasis; VEGFR-2 has been reported to be overexpressed and overstimulated in tumors and thus closely linked to the occurrence and development of multiple types of tumors (2,5,6). Targeting inhibition of VEGFR2 is a promising cancer therapeutic strategy based on multiple clinical trials (2,4,7). There is significant research and interest in the development of drugs targeting VEGFR2 inhibition (8) especially including dual inhibition in combination with other kinases or other proteins (9-15). 

References

  1. McTigue, M.A. et al. (1999) Structure 7:319.
  2. Liu, Y. et al. (2022) J. Hematol. Oncol. 15:89.
  3. Simons, M. et al. (2016) Nat. Rev. Mol. Cell Biol. 17:611.
  4. Yang, J. et al. (2018) Front. Immunol. 9:978.
  5. Zhao, J. et al. (2021) Drug Target. 29:155.
  6. Prasad, C.B. et al. (2022) Int. J. Biochem. Cell Biol. 142:106122.
  7. Zhu, P. et al. (2017) Onco. Targ. Ther. 10:4315.
  8. Shi, L. et al. (2016) Curr. Med. Chem. 23:1000.
  9. Chekler, E.L. et al. (2010) ACS Med. Chem. Lett. 1:488.
  10. Tang, Z. et al. (2016) Eur. J. Med. Chem. 118:328.
  11. Brands, R.C. et al. (2017) Oncol. Rep. 38:1877.
  12. Abdel-Mohsen, H.T. et al. (2019) Eur. J. Med. Chem. 179:707.
  13. Carvalho, B. et al. (2021) Sci. Rep. 11:6067. 
  14. Abd El-Meguid, E.A. et al. (2022) Bioorg. Med. Chem. Lett. 58:128529.
  15. Xue, X. et al. (2022) Invest. New Drugs. 40:10.

Long Name

Vascular Endothelial Growth Factor Receptor 2

Alternate Names

CD309, Flk-1, Flk1, KDR, KRD1, Ly73, VEGF R2

Entrez Gene IDs

3791 (Human); 16542 (Mouse)

Gene Symbol

KDR

UniProt

P35968.2

Additional VEGFR2/KDR/Flk-1 Products

Product Documents for Recombinant Human VEGFR2/KDR kinase domain His Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

Product Specific Notices for Recombinant Human VEGFR2/KDR kinase domain His Protein, CF

For research use only

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Protocols

View specific protocols for Recombinant Human VEGFR2/KDR kinase domain His Protein, CF (11740-KD):

Materials
  • Assay Buffer:  50 mM Tris, 20 mM MgCl2, 5 mM MnCl2, 0.1 mg/mL BSA, pH 7.5
  • Recombinant Human VEGFR2/KDR/Flk-1 kinase domain His-tag (rhVEGFR2) (Catalog # 11740-KD)
  • 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 (Promega)
  • White 96-well Plate
  • Plate Reader with Luminescence Read Capability
  1. Dilute rhVEGFR2 to 1.6 µg/mL in Assay Buffer.
  2. Prepare Substrate Mixture containing 200 µM ATP and 0.4 mg/mL Poly (4:1 Glu:Tyr) peptide in Assay Buffer.
  3. Combine equal volumes of 1.6 ng/µL rhVEGFR2 and Substrate Mixture. Create a Substrate Control by replacing enzyme with Assay Buffer.
  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-Glo 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:
  • rhVEGFR2: 0.8 μg/mL
  • ATP: 100 µM
  • Poly (4:1 Glu:Tyr) peptide: 0.2 mg/mL




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