Figure 1: Treatment of HUVE cells with VEGF stimulates phosphorylation of VEGFR-2 at Tyr1175, detected by PathScan® Phospho-VEGFR-2 (Tyr1175) Sandwich ELISA kit, #7335, but does not affect the level of total VEGFR-2 protein detected by PathScan® Total VEGFR-2 Sandwich ELISA kit, #7340. OD450 readings are shown in the top figure, while the corresponding western blot using Phospho-VEGFR-2 (Tyr1175) Rabbit mAb #2478 (right panel) or VEGFR-2 Rabbit mAb (7340-55B11) (left panel), is shown in the bottom figure.
Figure 2: Linear relationship between protein concentration of lysates from VEGF-treated HUVE cells and kit assay optical density readings. HUVE cells (85% confluence) were treated with VEGF (50 ng/ml), and lysed after growth at 37°C for 2 min.
|Product Includes||Volume||Solution Color|
|VEGFR-2 Mouse mAb Coated Microwells||96 tests|
|Phospho-VEGF Receptor 2 (Tyr1175) Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Phospho-VEGFR-2 (Tyr1175) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Phospho-VEGFR-2 (Tyr1175) protein. A VEGFR-2 Mouse mAb has been coated onto the microwells. After incubation with cell lysates, both nonphospho- and phospho-VEGFR-2 proteins are captured by the coated antibody. Following extensive washing, a phospho-VEGFR-2 Rabbit mAb is added to detect the captured phospho-VEGFR-2 protein. Anti-rabbit IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of phospho-VEGFR-2 protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Phospho-VEGFR-2 (Tyr1175) Sandwich ELISA Kit detects endogenous levels of Phospho-VEGFR-2 protein. Using this Sandwich ELISA Kit #7335, a significant induction of phospho-VEGFR-2 in HUVE* cells treated with VEGF can be detected. However, the level of total VEGFR-2 (phospho and non-phospho), detected by PathScan® Total VEGFR-2 Sandwich ELISA Kit #7340, remains unchanged (Figure 1). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
*Human Umbilical Vein Endothelial Cells
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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