The relationship between the protein concentration of lysate from untreated and VEGF-treated HUVE cells and the absorbance at 450 nM using PathScan® Total VEGFR-2 Sandwich ELISA Antibody Pair #7825 is shown. HUVE cells (85% confluence) were treated with VEGF (50 ng/ml) for 5 min. at 37ºC, and then lysed.
|7825S||1 Kit (Reagents for 4 x 96 well plates)||$ 469|
|Product Includes||Volume||Cap Color|
|VEGFR-2 Capture Mouse mAb (100X)||400 µl||Pink|
|VEGFR-2 Detection Rabbit mAb (100X)||400 µl||Blue|
|Anti-rabbit IgG, HRP-linked Antibody (1000X)||40 µl||Red|
Capture and detection antibodies are stored at 4°C. HRP-linked secondary reagent is stored at -20°C.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).
Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.
STOP Solution: (#7002)
NOTE: Reagents should be made fresh daily.
posted January 2008
revised Sepetember 2013
Protocol Id: 20
CST's PathScan® Total VEGFR-2 Sandwich ELISA Antibody Pair is being offered as an economical alternative to our PathScan® Total VEGFR-2 Sandwich ELISA Kit #7340. Capture and detection antibodies (100X stocks) and HRP-conjugated secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The VEGFR-2 capture antibody is coated on a 96 well microplate overnight in PBS. After blocking, cell lysates are added followed by a VEGFR-2 detection antibody and anti-rabbit IgG, HRP conjugated antibody. HRP substrate (TMB) is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of total VEGFR-2 protein.
For Antibody Pair specificity and sensitivity, please refer to the corresponding PathScan® Sandwich ELISA Kit. Note: This antibody pair detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Vascular endothelial growth factor receptor 2 (VEGFR2, KDR, Flk-1) is a major receptor for VEGF-induced signaling in endothelial cells. Upon ligand binding, VEGFR2 undergoes autophosphorylation and becomes activated (1). Major autophosphorylation sites of VEGFR2 are located in the kinase insert domain (Tyr951/996) and in the tyrosine kinase catalytic domain (Tyr1054/1059) (2). Activation of the receptor leads to rapid recruitment of adaptor proteins, including Shc, GRB2, PI3 kinase, NCK, and the protein tyrosine phosphatases SHP-1 and SHP-2 (3). Phosphorylation at Tyr1212 provides a docking site for GRB2 binding and phospho-Tyr1175 binds the p85 subunit of PI3 kinase and PLCγ, as well as Shb (1,4,5). Signaling from VEGFR2 is necessary for the execution of VEGF-stimulated proliferation, chemotaxis and sprouting, as well as survival of cultured endothelial cells in vitro and angiogenesis in vivo (6-8).
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