Phospho-KDR (Y951) Antibody

What is the specificity of Phospho-KDR (Y951) Antibody?

Phospho-KDR (Y951) antibody specifically detects endogenous levels of VEGFR2 (also known as KDR/Flk-1) only when phosphorylated at tyrosine 951. This antibody does not cross-react with other phosphorylation sites on VEGFR2 or with non-phosphorylated VEGFR2 . The specificity can be validated using phosphopeptide competition assays, where pre-incubation of the antibody with the phosphopeptide blocks detection of the signal in Western blotting and immunohistochemistry applications .

What applications is Phospho-KDR (Y951) Antibody validated for?

The antibody has been tested and validated in multiple experimental systems including human breast carcinoma tissues and human ovarian cancer cell lines (SK-OV3) .

What is the molecular weight of phosphorylated VEGFR2 in Western blot analysis?

While the predicted molecular weight of VEGFR2 is approximately 151 kDa, the mature form typically migrates at ~230 kDa in Western blot analysis due to extensive glycosylation of the extracellular domain . When using this antibody, researchers should look for bands at 230 kDa rather than at the theoretical molecular weight. This apparent molecular weight difference is an important consideration when interpreting Western blot results .

How should samples be prepared to effectively detect phosphorylated Y951 VEGFR2?

For optimal detection of phosphorylated Y951, samples should be prepared as follows:

• Cell stimulation: Serum-starve cells for 12-24 hours before stimulation with VEGF-A (typically 50-100 ng/ml for 5-10 minutes) .

• Lysis buffer composition: Use buffer containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride, and β-glycerophosphate) to preserve phosphorylation status .

• Sample handling: Process samples quickly and maintain at cold temperatures throughout to prevent dephosphorylation by endogenous phosphatases .

• Controls: Include both VEGF-stimulated and unstimulated samples, as well as phosphopeptide competition controls to validate specificity .

How can phosphopeptide blocking be used to validate antibody specificity?

Phosphopeptide blocking is a critical control experiment that validates the specificity of phospho-specific antibodies:

• Protocol design: Divide your sample into two aliquots. Pre-incubate the antibody with phosphopeptide (corresponding to the Y951 region) for one aliquot, while using the antibody directly with the other .

• Expected results: The phosphopeptide should compete for antibody binding, resulting in loss or significant reduction of signal in Western blot, IHC, or IF applications .

• Interpretation: As demonstrated in the validation images for multiple commercial antibodies, the signal should be present in untreated samples but absent or significantly reduced in the phosphopeptide-treated condition .

• Quantification: The degree of signal reduction (typically >90%) provides evidence for the specificity of the antibody .

What is the biological significance of VEGFR2 Y951 phosphorylation in endothelial cell function?

Y951 phosphorylation plays distinct roles in endothelial cell biology:

• Cell migration specificity: Phosphorylation at Y951 and its coupling to the adapter molecule TSAd (T-cell specific adapter) is critical for VEGF-A-induced organization of the actin cytoskeleton and endothelial cell migration, but not for proliferation .

• Experimental evidence: Studies using phosphorylation-blocking peptides and TSAd-specific siRNA demonstrated that disruption of Y951 phosphorylation or TSAd expression inhibits VEGF-A-induced actin stress fiber formation and cell motility without affecting DNA synthesis .

• Pericyte association: Endothelial cells with VEGFR2 phosphorylated at Y951 typically lack associated pericytes, while vessels without Y951 phosphorylation are more often covered by pericyte-like, α-smooth muscle actin-positive cells .

• Signaling pathway: Y951 phosphorylation mediates complex formation between TSAd and Src, suggesting a mechanism for regulating endothelial cell migration through cytoskeletal reorganization .

How does Y951 phosphorylation differ from other VEGFR2 phosphorylation sites?

VEGFR2 contains multiple phosphorylation sites with distinct functions:

Research strategies to distinguish the roles of different phosphorylation sites include:

• Site-specific phospho-antibodies for detection

• Y-to-F mutations in VEGFR2 to prevent specific site phosphorylation

• Phosphomimetic mutations (Y-to-D/E) to simulate constitutive phosphorylation

• Phosphorylation-blocking peptides that compete with specific sites

What experimental approaches can determine the temporal dynamics of Y951 phosphorylation?

To investigate temporal dynamics of Y951 phosphorylation:

• Time-course experiments: Stimulate cells with VEGF-A and collect samples at intervals (0, 2, 5, 10, 30, 60, 120 minutes) to analyze the kinetics of phosphorylation .

• Phosphopeptide transfection: As demonstrated in the literature, cells can be transfected with phospho-Y951 peptides, with phosphorylation remaining detectable for approximately 120 minutes, allowing analysis of long-term responses like DNA synthesis .

• Live-cell imaging: Using fluorescently tagged biosensors designed to detect conformational changes upon Y951 phosphorylation.

• Phosphoproteomics: Mass spectrometry-based approaches can quantitatively assess phosphorylation changes at multiple sites simultaneously.

Why might background staining occur when using Phospho-KDR (Y951) Antibody?

Background staining can result from several factors:

• Non-specific binding: Use appropriate blocking agents (5% BSA or 5% non-fat milk) in TBS-T buffer. For tissues with high endogenous biotin, use avidin-biotin blocking kits .

• Insufficient washing: Extend washing steps (3-5 washes of 5-10 minutes each) with TBS-T to reduce background .

• Antibody concentration: Optimize dilution through titration experiments. Starting recommendations are 1:500-1:2000 for WB and 1:100-1:300 for IHC .

• Fixation artifacts: For IF/IHC, compare different fixation methods (paraformaldehyde, methanol, acetone) to determine optimal conditions for phospho-epitope preservation .

• Endogenous phosphatase activity: Ensure phosphatase inhibitors are fresh and used at appropriate concentrations in all buffers until the fixation step is complete .

How can I distinguish between specific Y951 phosphorylation and cross-reactivity with other phospho-sites?

To ensure detection specificity:

• Phosphopeptide competition: As a definitive control, pre-incubate the antibody with phospho-Y951 peptide. If signal disappears, it confirms specificity .

• Dephosphorylation controls: Treat duplicate samples with lambda phosphatase to remove all phosphorylation; signal should disappear with phospho-specific antibodies.

• Y951F mutants: In overexpression systems, compare wild-type VEGFR2 with Y951F mutant; the mutant should show no signal with phospho-Y951 antibody after VEGF stimulation .

• Multiple antibody validation: Use antibodies from different sources targeting the same phospho-site and compare staining patterns .

How can Phospho-KDR (Y951) Antibody be used to study tumor angiogenesis?

Phospho-KDR (Y951) antibody offers several approaches to investigate tumor angiogenesis:

• Tumor tissue analysis: IHC or IF staining of tumor sections can reveal patterns of Y951 phosphorylation in tumor vasculature compared to normal vessels .

• Correlation with pericyte coverage: Dual staining with pericyte markers (α-SMA) and phospho-Y951 can assess vessel maturation status, as vessels lacking Y951 phosphorylation more often have pericyte coverage .

• Drug efficacy assessment: Monitor changes in Y951 phosphorylation to evaluate the efficacy of anti-angiogenic therapies targeting VEGF/VEGFR2 signaling .

• Tumor models: In xenograft or genetically engineered mouse models, assess how Y951 phosphorylation correlates with tumor growth, metastasis, and response to therapy .

• Mechanistic studies: Investigate how tumor microenvironment factors influence Y951 phosphorylation and subsequent TSAd-dependent migration of endothelial cells .

What insights does VEGFR2 Y951 phosphorylation provide about vascular development and disease?

Y951 phosphorylation status offers valuable insights into vascular biology:

• Vessel maturation: The inverse relationship between Y951 phosphorylation and pericyte coverage suggests Y951 phosphorylation is prominent during active angiogenesis but reduced in mature vessels .

• Differential phosphorylation: Studies in embryoid body models showed that while Y1175 and Y1214 phosphorylation occurs in most VEGFR2-expressing vessels, Y951 phosphorylation is more selectively present, suggesting context-dependent regulation .

• Pathological angiogenesis: The selective presence of Y951 phosphorylation makes it a potential biomarker to distinguish pathological from physiological angiogenesis .

• Therapeutic implications: The specific role of Y951 in migration but not proliferation suggests that targeting this pathway could inhibit vessel sprouting while potentially preserving other VEGFR2 functions .