Phospho-KDR/FLT4 (Y1054/Y1063) Antibody

Basic Research Questions

• What is the molecular significance of Y1054/Y1063 phosphorylation in KDR/FLT4 receptors?

  Phosphorylation at Y1054 and Y1059/Y1063 (the exact numbering may vary between resources) is critical for the kinase activity of VEGFR2/KDR. These phosphorylation sites are located in the kinase domain activation loop, which regulates receptor tyrosine kinase function . The phosphorylation of these tyrosine residues is a key step in VEGF signaling pathways that promote angiogenesis, endothelial cell proliferation, migration, and survival.

  According to research, phosphorylation at Y1054 and Y1059 may be preceded by autophosphorylation at Y801, initiating a cascade of phosphorylation events . This activation mechanism is essential for the proper functioning of these receptors in both physiological and pathological angiogenesis.

• What are the optimal experimental conditions for using Phospho-KDR/FLT4 (Y1054/Y1063) antibodies in Western blotting?

  For optimal Western blot results with Phospho-KDR/FLT4 (Y1054/Y1063) antibodies, follow these methodological guidelines:

  • Dilution: Use 1:500 to 1:1000 dilution for Western blot applications

  • Molecular Weight: Expect to detect a band at approximately 230 kDa, although the calculated molecular weight is around 151-152 kDa

  • Buffer Conditions: Use PVDF membrane with appropriate Western blot buffer (e.g., Buffer Group 1 for some antibodies)

  • Sample Preparation: For positive controls, use pervanadate-treated cells (e.g., 100 μM for 10 minutes)

  • Detection System: HRP-conjugated secondary antibodies work well with these primary antibodies

  When troubleshooting, verify phosphorylation status by comparing untreated versus treated samples, as demonstrated in control experiments showing clear differential detection .

• How can I validate the specificity of my Phospho-KDR/FLT4 (Y1054/Y1063) antibody?

  Validating antibody specificity is critical for reliable research results. Follow these methodological approaches:

  • Positive Controls: Use cell lines known to express phosphorylated VEGFR2/3, such as A431 human epithelial carcinoma cells treated with pervanadate

  • Comparison Tests: Run parallel Western blots with phosphorylated and non-phosphorylated samples to confirm specificity

  • Peptide Competition: Perform blocking experiments with the specific phosphopeptide used as the immunogen

  • Phosphatase Treatment: Treat half of your sample with lambda phosphatase to remove phosphorylation and confirm loss of signal

  • Knockout/Knockdown Validation: If possible, use VEGFR2/3 knockout or knockdown samples as negative controls

  The immunogen typically used for these antibodies is a synthesized peptide derived from human Flk-1/Flt-4 around the phosphorylation site of Y1054/Y1063 , making synthetic phosphopeptides useful for validation experiments.

• What applications are most suitable for Phospho-KDR/FLT4 (Y1054/Y1063) antibodies?

  Based on the search results, these antibodies have been validated for several applications:

  Application	Recommended Dilution	Notes
  Western Blot (WB)	1:500-1:1000	Most commonly validated application
  Immunohistochemistry (IHC)	1:50-1:300	Works on both paraffin and frozen sections
  ELISA	1:40000	High sensitivity application
  Immunofluorescence (IF)	1:50-1:200	For cellular localization studies

  When using these antibodies for IHC applications, heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0) is recommended for optimal results .