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12872S 100 µl (50 tests) $299.00
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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Endogenous 210,230 Rabbit IgG
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Confocal immunofluorescent analysis of rat pancreas using VEGF Receptor 2 (D5B1) Rabbit mAb (Alexa Fluor® 555 Conjugate) (red) and β-Catenin (L54E2) Mouse mAb (IF Preferred) #2677 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Immunofluorescence (Frozen)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
  5. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Frozen/Cryostat Sections (IF-F)

  1. For fixed frozen tissue proceed with Immunostaining (Section C).
  2. For fresh, unfixed frozen tissue, please fix immediately, as follows:
    1. Cover sections with 4% formaldehyde dilute in 1X PBS.

      NOTE: Formaldehyde is toxic, use only in fume hood.

    2. Allow sections to fix for 15 minutes at room temperature.
    3. Aspirate liquid, rinse three times in 1X PBS for 5 minutes each.
    4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  7. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

protocol id: 222

Product Usage Information

Application Dilutions
Immunofluorescence 1:50

Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Specificity / Sensitivity

VEGF Receptor 2 (D5B1) Rabbit mAb (Alexa Fluor® 555 Conjugate) recognizes endogenous levels of total VEGF receptor 2 protein.


Species Reactivity: Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a recombinant protein containing the carboxy-terminal 150 amino acid residues of human VEGF receptor 2 protein.

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 555 fluorescent dye and tested in-house for immunofluorescent analysis in rat cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated VEGF Receptor 2 (D5B1) Rabbit mAb #9698.


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.


Entrez-Gene Id 3791
Swiss-Prot Acc. P35968


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
DRAQ5® is a registered trademark of Biostatus Limited.
The Alexa Fluor® dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.