Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total VEGFR-2 Sandwich ELISA Kit #7340

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Kit Includes Volume Solution Color
VEGFR-2 Mouse Antibody coated Microwells 96 well plate
VEGFR-2 Rabbit Monoclonal Detection Antibody 11 ml Green
Anti-rabbit IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

CST's PathScan® Total VEGFR-2 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total VEGFR-2 protein. A VEGFR-2 Mouse mAb (7335-1D6*) 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 VEGFR-2 Rabbit mAb (7340-55B11*) is added to detect the captured VEGFR-2 protein. HRP-linked anti-rabbit antibody (#7074*) 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 total VEGFR-2 protein.* Antibodies in this kit are custom formulations specific to the kit.

Specificity / Sensitivity

CST's PathScan® Total VEGFR-2 Sandwich ELISA Kit detects endogenous levels of total VEGFR-2 protein. Using PathScan® Phospho-VEGFR-2 (Tyr1175) 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 this 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

ELISA - Western correlation

ELISA - Western correlation

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.

Sensitivity

Sensitivity

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.

Background

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).

  1. Meyer, M. et al. (1999) EMBO J 18, 363-74.
  2. Dougher-Vermazen, M. et al. (1994) Biochem Biophys Res Commun 205, 728-38.
  3. Kroll, J. and Waltenberger, J. (1997) J Biol Chem 272, 32521-7.
  4. Takahashi, T. et al. (2001) EMBO J 20, 2768-78.
  5. Holmqvist, K. et al. (2004) J Biol Chem 279, 22267-75.
  6. Karkkainen, M.J. and Petrova, T.V. (2000) Oncogene 19, 5598-605.
  7. Rahimi, N. et al. (2000) J Biol Chem 275, 16986-92.
  8. Claesson-Welsh, L. (2003) Biochem Soc Trans 31, 20-4.

Application References

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Protocols

Companion Products

Rabbit Monoclonals Produced UsingTechnology from Epitomics, Inc. UnderU.S. patent No. 5,675,063.

For Research Use Only. Not For Use In Diagnostic Procedures.

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