KDR (Ab-951) Antibody

What is KDR (Ab-951) Antibody and what epitope does it recognize?

KDR (Ab-951) Antibody is a rabbit polyclonal antibody that specifically recognizes the VEGF Receptor 2 (VEGFR2/KDR) when phosphorylated at tyrosine residue 951 (Tyr951). The antibody targets a specific peptide sequence around amino acids 949-953 (K-D-Y-V-G) derived from human VEGFR2. This antibody is valuable for detecting endogenous VEGFR2 only when this specific tyrosine residue is phosphorylated, making it useful for studying VEGFR2 activation states .

What species reactivity has been confirmed for KDR (Ab-951) Antibody?

KDR (Ab-951) Antibody has confirmed reactivity with human, mouse, and rat specimens. This cross-species reactivity makes it valuable for comparative studies across different model systems. Researchers should validate optimal dilutions when working with each species as sensitivity may vary slightly across species despite the high sequence conservation in the target region .

What are the storage and handling recommendations for maintaining KDR (Ab-951) Antibody activity?

The antibody should be stored at -20°C or -80°C for long-term storage. It is supplied at 1.0mg/mL in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, containing 150mM NaCl, 0.02% sodium azide, and 50% glycerol as stabilizers. Researchers should avoid repeated freeze-thaw cycles as these can degrade antibody performance. For short-term storage (up to 6 months), the antibody can be kept at 4°C. Always centrifuge briefly before opening the vial to ensure solution homogeneity .

What are the validated applications for KDR (Ab-951) Antibody and their recommended dilutions?

KDR (Ab-951) Antibody has been validated for multiple research applications with specific recommended dilutions:

These dilutions serve as starting points and may require optimization based on sample type, preparation method, and detection system .

How should researchers design controls when using KDR (Ab-951) Antibody for phosphorylation studies?

For phosphorylation studies with KDR (Ab-951) Antibody, researchers should implement multiple controls:

• Positive control: Cells treated with VEGF to stimulate VEGFR2 phosphorylation

• Negative control: Untreated cells or cells treated with VEGFR2 kinase inhibitors

• Phosphatase control: Sample treated with lambda phosphatase to demonstrate phospho-specificity

• Loading control: Detection of total VEGFR2 using a non-phospho-specific antibody

• Peptide competition: Pre-incubation with phospho-peptide (K-D-pY-V-G) should abolish signal

These controls help validate that observed signals represent specific detection of phosphorylated Tyr951 on VEGFR2 rather than non-specific binding or artifacts .

What are the recommended sample preparation protocols for optimal KDR (Ab-951) Antibody performance?

For optimal results with KDR (Ab-951) Antibody, sample preparation should preserve phosphorylation status:

• Cell lysis: Use buffers containing phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride, β-glycerophosphate)

• Tissue samples: Flash-freeze immediately after collection and homogenize in ice-cold lysis buffer with phosphatase inhibitors

• Fixation for IHC/IF: 4% paraformaldehyde is preferred; avoid prolonged fixation

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Blocking: Use 5% BSA rather than milk (which contains phosphatases) in TBS-T

These methodological considerations help maintain phospho-epitope integrity throughout the experimental workflow .

How does KDR (Ab-951) Antibody performance compare with other VEGFR2 phospho-site specific antibodies in angiogenesis research?

KDR (Ab-951) Antibody specifically detects phosphorylation at Tyr951, which is particularly important for understanding VEGFR2 signaling in angiogenesis. The Tyr951 residue serves as a binding site for TSAd (T-cell-specific adaptor) and contributes to VEGF-induced actin reorganization and migration, distinct from other phosphorylation sites:

• pTyr951: Primarily involved in cell migration and vascular permeability pathways

• pTyr1054/1059: Critical for kinase activation

• pTyr1175: Main site for PLCγ binding and proliferation signaling

• pTyr1214: Involved in p38 MAPK activation

When investigating specific aspects of VEGFR2 signaling, researchers should select phospho-antibodies targeting the relevant sites. For comprehensive pathway analysis, consider using multiple phospho-site specific antibodies in parallel experiments .

What are the potential cross-reactivity concerns with KDR (Ab-951) Antibody in complex tissue samples?

While KDR (Ab-951) Antibody demonstrates high specificity for phosphorylated VEGFR2 at Tyr951, researchers should be aware of potential cross-reactivity issues in complex tissue samples:

• Similar phospho-tyrosine motifs in related receptor tyrosine kinases (RTKs)

• Non-specific binding to denatured proteins in fixed tissues

• Background in highly vascularized tissues with abundant VEGFR2 expression

To address these concerns:

• Always include appropriate negative controls

• Validate antibody specificity using siRNA/shRNA knockdown of VEGFR2

• Consider peptide competition assays with phospho-Tyr951 peptide

• Use dual staining approaches with other VEGFR2 antibodies to confirm specificity

For particularly challenging samples, consider using phospho-enrichment techniques prior to analysis to enhance signal-to-noise ratio .

How can researchers optimize KDR (Ab-951) Antibody for detecting low-abundance phosphorylation events?

Detecting low-abundance phosphorylation events requires optimization strategies:

• Signal amplification: Use tyramide signal amplification (TSA) for IHC/IF or highly sensitive ECL substrates for WB

• Sample enrichment: Perform immunoprecipitation of total VEGFR2 before probing with phospho-specific antibody

• Phosphatase inhibition: Use cocktails containing both serine/threonine and tyrosine phosphatase inhibitors

• Stimulation conditions: Optimize VEGF concentration and stimulation time to maximize phosphorylation

• Quantitative techniques: Consider using phospho-flow cytometry for single-cell analysis

These approaches can significantly improve detection sensitivity, especially in primary cells or tissue samples where phosphorylation signals may be transient or weak .

How can KDR (Ab-951) Antibody be incorporated into multiplexed phospho-signaling analysis workflows?

KDR (Ab-951) Antibody can be integrated into multiplexed phospho-signaling workflows to understand VEGFR2 pathway dynamics:

• Sequential immunoblotting: Strip and reprobe membranes with antibodies for downstream mediators (PLCγ, ERK, AKT)

• Multiplexed immunofluorescence: Combine with other phospho-antibodies using spectrally distinct fluorophores

• Mass cytometry (CyTOF): Conjugate with metal isotopes for high-dimensional single-cell analysis

• Proximity ligation assay (PLA): Pair with antibodies against interaction partners to detect molecular proximity

• Reverse phase protein array (RPPA): Profile multiple phosphorylation events in parallel

When designing multiplexed experiments, select antibodies raised in different host species to avoid cross-reactivity during detection, and carefully validate each antibody individually before combining them .

What are the considerations for using KDR (Ab-951) Antibody in quantitative phosphoproteomics studies?

When incorporating KDR (Ab-951) Antibody into phosphoproteomics workflows:

• Antibody-based enrichment: Use the antibody for immunoprecipitation before mass spectrometry analysis

• Validation of MS findings: Confirm phosphoproteomics hits using the antibody in orthogonal assays

• Quantification standards: Include phosphopeptide standards for the Tyr951 epitope for calibration

• Spatial information: Complement MS data with IF/IHC to provide spatial context for phosphorylation events

• Temporal dynamics: Use the antibody to validate temporal phosphorylation profiles identified in MS studies

This integration provides complementary advantages: the specificity of antibody-based detection and the comprehensive coverage of mass spectrometry-based phosphoproteomics .

How can researchers use KDR (Ab-951) Antibody to investigate VEGFR2 signaling in pathological angiogenesis models?

For investigating pathological angiogenesis, researchers can apply KDR (Ab-951) Antibody in several specialized approaches:

• Tumor xenograft models: Assess phospho-VEGFR2 (Tyr951) in tumor vasculature after anti-angiogenic treatments

• Retinal neovascularization models: Quantify phospho-VEGFR2 in retinal whole-mounts

• Ex vivo angiogenesis assays: Monitor phosphorylation dynamics in aortic ring sprouting assays

• Patient-derived samples: Compare phospho-VEGFR2 patterns in normal versus pathological tissue samples

• Drug screening: Evaluate compound effects on VEGFR2 phosphorylation status

The VEGFR2 signaling system plays a key role in vascular development and regulation of vascular permeability. In pathological contexts like HIV-1 infection, interaction with extracellular viral Tat protein can enhance angiogenesis in Kaposi's sarcoma lesions, making phospho-specific detection particularly valuable in understanding disease mechanisms .

How is KDR (Ab-951) Antibody being utilized in research on VEGFR2 inhibitor resistance mechanisms?

Researchers investigating resistance to VEGFR2-targeted therapies are using KDR (Ab-951) Antibody to:

• Monitor phosphorylation status at Tyr951 versus other sites (Tyr1175, Tyr1214) during treatment

• Identify differential phosphorylation patterns in resistant versus sensitive tumors

• Study compensatory signaling through alternative phosphorylation sites

• Evaluate combination treatments targeting multiple RTK pathways

• Develop phosphorylation-based biomarkers for predicting treatment response

This application helps elucidate molecular mechanisms of resistance to anti-angiogenic therapies by determining whether resistance involves bypass of VEGFR2 signaling or altered phosphorylation patterns within the receptor itself .

What considerations should researchers make when using KDR (Ab-951) Antibody in single-cell signaling analysis?

For single-cell analysis of VEGFR2 phosphorylation:

• Fixation optimization: Develop protocols that preserve phospho-epitopes while maintaining cellular architecture

• Signal amplification: Implement techniques like tyramide signal amplification for detecting low-abundance signals

• Multiplexed detection: Combine with markers for cell identity, cell cycle, and downstream signaling

• Image analysis: Use automated image analysis tools to quantify phospho-VEGFR2 intensity at single-cell resolution

• Validation approaches: Confirm findings with orthogonal techniques like phospho-flow cytometry

These approaches allow researchers to address heterogeneity in VEGFR2 signaling across different cell populations within complex tissues, providing insights into differential responses to angiogenic stimuli or inhibitors .

What are common challenges and solutions when using KDR (Ab-951) Antibody in Western blotting applications?

Researchers may encounter several challenges when using this antibody for Western blotting:

When working with phospho-specific antibodies like KDR (Ab-951), sample preparation is critical for maintaining phosphorylation status throughout the experimental procedure .

How can researchers verify the specificity of KDR (Ab-951) Antibody detection in their experimental system?

To verify antibody specificity:

• Phosphatase treatment: Treating duplicate samples with lambda phosphatase should eliminate signal

• Peptide competition: Pre-incubating antibody with phospho-peptide versus non-phospho-peptide

• Genetic approaches: Testing samples from VEGFR2 knockout models or after siRNA knockdown

• Pharmacological inhibition: Using selective VEGFR2 kinase inhibitors to block phosphorylation

• Mass spectrometry validation: Confirming phosphorylation status of Tyr951 in samples showing positive signal

These validation steps are essential for ensuring that observed signals genuinely represent phosphorylated VEGFR2 at Tyr951 rather than artifacts or cross-reactivity with other phospho-proteins .

How does the performance of polyclonal KDR (Ab-951) Antibody compare with monoclonal alternatives for research applications?

The polyclonal KDR (Ab-951) Antibody offers distinct advantages and limitations compared to monoclonal alternatives:

For critical quantitative studies or clinical research applications, researchers may benefit from comparing results using both antibody types. For qualitative detection of phosphorylated VEGFR2, the polyclonal antibody often provides sufficient specificity with enhanced sensitivity .

What complementary research tools should be considered alongside KDR (Ab-951) Antibody for comprehensive VEGFR2 pathway analysis?

For comprehensive VEGFR2 pathway analysis, consider these complementary approaches:

• Total VEGFR2 antibodies: To normalize phospho-signal to total receptor expression

• Antibodies against other phosphorylation sites (pTyr1054/1059, pTyr1175, pTyr1214)

• Downstream signaling markers: Phospho-PLCγ, phospho-ERK, phospho-AKT

• VEGF ligand detection tools: ELISA kits or antibodies for quantifying VEGF levels

• Functional assays: Endothelial cell migration, tube formation, and proliferation assays

This multi-faceted approach enables researchers to correlate receptor phosphorylation status with downstream signaling events and functional outcomes, providing a more complete picture of VEGFR2 pathway activation in their experimental system .

How might KDR (Ab-951) Antibody contribute to research on emerging targeted therapies for VEGFR2-dependent diseases?

KDR (Ab-951) Antibody can advance research on targeted therapies by:

• Elucidating site-specific phosphorylation patterns in response to novel inhibitors

• Identifying differential effects of inhibitors on distinct VEGFR2 phosphorylation sites

• Monitoring treatment response in patient-derived samples or xenograft models

• Characterizing resistance mechanisms involving altered phosphorylation patterns

• Developing phosphorylation-based companion diagnostics for therapy selection

The VEGF-kinase ligand/receptor signaling system plays a crucial role in vascular development and regulation of vascular permeability, making it a key target for therapeutic intervention in multiple disease contexts .

What emerging technologies might enhance the utility of KDR (Ab-951) Antibody in future research applications?

Emerging technologies that will enhance KDR (Ab-951) Antibody applications include: