Human vegfr2

Manufactured by R&D Systems

Sourced in United States

Human VEGFR2 is a recombinant protein that represents the extracellular domain of the human vascular endothelial growth factor receptor 2 (VEGFR2). VEGFR2 is a receptor tyrosine kinase that plays a crucial role in angiogenesis and vascular development.

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Lab products found in correlation

Superdex 75 increase, by GE Healthcare (1 mentions) Sec uplc, by Waters Corporation (1 mentions) 293 expi cells, by Thermo Fisher Scientific (1 mentions) Freestyle max 293 expression system, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

VEGFR2 (23 citations) Human VEGFR2/KDR Quantikine ELISA Kit (2 citations) Human VEGFR2 ELISA kit (2 citations)

2 protocols using human vegfr2

Isolation and Purification of VEGFR2-Binding Fab

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KD035 was identified by panning of Dyax FAB‐310 phagemid library on immobilized human VEGFR2 (R&D systems) followed by affinity maturation using a light chain shuffling method. The constructs carrying KD035 antigen‐binding fragment (Fab) domain were cloned into the mammalian expression vector pBh1 (Dyax) and transiently expressed in human 293 Expi cells (Invitrogen). Fabs were purified from cell culture supernatant by passing it several times through a protein A Sepharose HP column (GE Healthcare) using a peristaltic pump for a period of 8 h and eluting with 1 M Glycine pH 2.0. The eluate was further purified by size exclusion chromatography (SEC) using a Superdex 75 increase (GE Healthcare) column.
The variable domain amino acid sequence of IMC‐1121B (which shares the amino acid sequence of ramucirumab as described in US 8057791 B2/EP 1916001 A2) was reverse‐translated into DNA sequences and optimized by using Integrated DNA Technologies tools. The synthesized genes of 1121 light chain and heavy chain variable domains were cloned into a mammalian expression vector pBh1 (Dyax). The plasmid DNA containing 1121 gene was transfected and expressed by using the FreeStyle™ MAX 293 Expression System (ThermoFisher Scientific) according to the manufacture instruction. The supernatant was harvested and purified by protein A (GE) with at least 95% monomer detected by SEC‐UPLC (Waters).

Depetris R.S., Lu D., Polonskaya Z., Zhang Z., Luna X., Tankard A., Kolahi P., Drummond M., Williams C., Ebert M.C., Patel J.P, & Poyurovsky M.V. (2021). Functional antibody characterization via direct structural analysis and information‐driven protein–protein docking. Proteins, 90(4), 919-935.

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VEGFR2 and EphA2 ELISA Assays

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Ninety six-well ELISA high binding plates (Costar, Corning, NY, USA) were incubated overnight with 100 μl/well of 0.5 µg/ml human VEGFR2 (R&D Systems). Then, plates were washed three times with PBS + 0.05% tween 20, pH 7.5, and blocked by a 1 h incubation at 37 °C with PBS + 1% BSA. After washing, increasing concentrations of UniPR1331 were added and preincubated for 1 h. Then, human biotinylated VEGF-165-Fc (300 ng/ml) was added and incubated for 4 h at 37 °C. After washing, streptavidin-HRP (0.05 μg/ml in PBS + 1% BSA) was added and incubated for 1 h at room temperature. After further washing, stable peroxide buffer pH 5.0 [11.3 g/l citric acid, 9.7 g/l sodium phosphate, 0.02% H₂O₂ (30%, m/m, in water)] containing 0.1 mg/ml tetramethylbenzidine was added and the colorimetric reaction was allowed to occur. The reaction was stopped with 3 N HCl and the absorbance at 450 nm was measured using an ELISA plate reader (Sunrise, TECAN, Switzerland). ELISA assays on EphA2 were performed basically as described for VEGFR2. For more details, see Tognolini et al. [25 (link)].

Rusnati M., Paiardi G., Tobia C., Urbinati C., Lodola A., D’Ursi P., Corrado M., Castelli R., Wade R.C., Tognolini M, & Chiodelli P. (2021). Cholenic acid derivative UniPR1331 impairs tumor angiogenesis via blockade of VEGF/VEGFR2 in addition to Eph/ephrin. Cancer Gene Therapy, 29(7), 908-917.

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