KDR (Ab-1214) Antibody

What is VEGFR2/KDR and why is it significant in research?

VEGFR2 (Vascular Endothelial Growth Factor Receptor 2), also known as KDR (Kinase Insert Domain Receptor) or Flk-1, is a tyrosine-protein kinase receptor that plays a critical role in vascular development and regulation of vascular permeability. This receptor has a molecular weight of approximately 230 kDa and functions as a key mediator in the VEGF signaling pathway . VEGFR2 is particularly important in research related to angiogenesis, tumor development, and cardiovascular diseases. In HIV-1 infection contexts, the interaction between VEGFR2 and extracellular viral Tat protein appears to enhance angiogenesis in Kaposi's sarcoma lesions . The receptor's central role in these physiological and pathological processes makes it an important target for both basic research and therapeutic development.

What epitope does the KDR (Ab-1214) Antibody recognize?

The KDR (Ab-1214) Antibody specifically recognizes a peptide sequence around amino acids 1212~1216 (F-H-Y-D-N) derived from Human VEGFR2 . This epitope is located in the cytoplasmic domain of the receptor. It's important to note that this antibody detects the endogenous level of total VEGFR2 protein regardless of phosphorylation status . This differs from phospho-specific antibodies such as Human Phospho-VEGFR2/KDR/Flk-1 (Y1214) Antibody, which specifically recognizes VEGFR2 when phosphorylated at tyrosine 1214 .

What applications is the KDR (Ab-1214) Antibody validated for?

Based on technical documentation, the KDR (Ab-1214) Antibody has been validated for the following applications :

The antibody has been successfully used to detect VEGFR2 in human breast carcinoma tissue by IHC and in methanol-fixed MCF cells by immunofluorescence , demonstrating its utility in both tissue and cellular applications.

How should I store and handle the KDR (Ab-1214) Antibody?

The KDR (Ab-1214) Antibody should be stored at -20°C or -80°C upon receipt . It is critical to avoid repeated freeze-thaw cycles as these can degrade the antibody and reduce its binding efficiency. The antibody is supplied at a concentration of 1.0 mg/mL in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, with 0.02% sodium azide and 50% glycerol as preservatives .

For optimal performance:

• Aliquot the antibody into smaller volumes upon first thaw to minimize freeze-thaw cycles

• When thawing, allow the antibody to equilibrate to room temperature slowly

• Keep the antibody on ice when in use

• Return to -20°C or -80°C promptly after use

• Monitor storage conditions carefully, as antibody stability is critical for reproducible results

What controls should I include when using KDR (Ab-1214) Antibody?

When designing experiments with KDR (Ab-1214) Antibody, the following controls should be incorporated to ensure reliable and interpretable results:

• Positive control: Use samples known to express VEGFR2, such as human breast carcinoma tissue or A431 human epithelial carcinoma cell line (especially when treated with pervanadate for phosphorylation studies)

• Blocking peptide control: The search results demonstrate that preincubation with a blocking peptide eliminates specific staining, confirming antibody specificity

• Negative controls:

  • Secondary antibody-only control (omitting primary antibody)

  • Isotype control (non-specific antibody of the same isotype)

  • Samples known not to express VEGFR2 or where VEGFR2 has been knocked down

• Treatment controls: For phosphorylation studies, include both untreated and treated samples (e.g., pervanadate treatment for phospho-VEGFR2 detection)

Including these controls enables discrimination between specific signal and background or non-specific binding, particularly important for a polyclonal antibody like KDR (Ab-1214).

How can I distinguish between total VEGFR2 and phosphorylated VEGFR2 in my research?

Distinguishing between total and phosphorylated VEGFR2 requires careful experimental design and appropriate antibody selection:

• Detection approach:

  • KDR (Ab-1214) Antibody detects total VEGFR2 regardless of phosphorylation status

  • Phospho-specific antibodies (e.g., Human Phospho-VEGFR2/KDR/Flk-1 (Y1214) Antibody) detect only VEGFR2 molecules phosphorylated at specific residues

• Experimental strategy:

  • Run parallel samples on separate blots, one probed with KDR (Ab-1214) Antibody and another with phospho-specific antibody

  • Alternatively, strip and reprobe a single membrane (though this may reduce sensitivity)

  • Calculate the ratio of phospho-VEGFR2 to total VEGFR2 to assess activation status

• Sample preparation:

  • For phospho-VEGFR2 detection, samples often require treatment with activators like pervanadate, as demonstrated in the search results

  • Phosphatase inhibitors must be included in all buffers to preserve phosphorylation status

  • Process samples quickly to prevent dephosphorylation

• Data interpretation:

  • An increase in phospho-VEGFR2 without changes in total VEGFR2 indicates receptor activation

  • Changes in total VEGFR2 may reflect altered expression, degradation, or trafficking

This dual detection approach provides comprehensive information about both VEGFR2 expression levels and activation state, which is critical for signaling pathway studies.

What are the considerations for using KDR (Ab-1214) Antibody in cancer research models?

Using KDR (Ab-1214) Antibody in cancer research requires attention to several key factors:

• Model selection considerations:

  • Cell line heterogeneity: VEGFR2 expression varies significantly between cancer cell lines

  • Animal models: Species reactivity (confirmed for human and mouse ) must be considered

  • Patient-derived samples: Fixation methods may affect epitope preservation

• Technical optimization:

  • Antigen retrieval methods for FFPE tissues should be optimized (the antibody has been validated on paraffin-embedded human breast carcinoma tissue )

  • Signal amplification systems may be needed for low-expressing samples

  • Co-staining with endothelial markers helps distinguish tumor cell vs. endothelial VEGFR2 expression

• Experimental design:

  • Include treatment time courses to capture dynamic VEGFR2 changes

  • Consider both membrane and cytoplasmic staining when analyzing results

  • Correlate VEGFR2 expression with functional angiogenesis assays (e.g., tube formation)

• Contextual analysis:

  • Assessment of other angiogenic markers alongside VEGFR2

  • Correlation with clinical parameters in patient samples

  • Evaluation of VEGFR2 in relation to hypoxic regions in tumors

The antibody's validation in breast carcinoma tissue provides a foundation for these cancer research applications, though protocol optimization may be needed for different cancer types.

How does the polyclonal nature of KDR (Ab-1214) Antibody affect experimental results?

The polyclonal nature of KDR (Ab-1214) Antibody has several important implications for experimental design and data interpretation:

These characteristics make polyclonal antibodies like KDR (Ab-1214) valuable for detection and initial characterization, though highly specific applications may require additional validation.

How can I validate KDR (Ab-1214) Antibody specificity for my specific research application?

Thorough validation of KDR (Ab-1214) Antibody specificity is essential for reliable research outcomes and should include:

• Epitope blocking experiments:

  • Pre-incubate the antibody with blocking peptide before application

  • This approach effectively eliminates specific staining as demonstrated in the immunohistochemical analysis of human breast carcinoma tissue

• Genetic validation approaches:

  • Use VEGFR2 knockout or knockdown samples as negative controls

  • Compare staining in cells with known differential expression of VEGFR2

  • Consider overexpression systems for positive controls

• Application-specific validation:

  • For Western blot: Confirm single band at expected molecular weight (~230 kDa)

  • For IHC/IF: Compare staining pattern with published literature

  • For ELISA: Create standard curves with recombinant VEGFR2 protein

• Cross-validation with orthogonal methods:

  • Confirm protein expression with alternative detection methods (mass spectrometry, other validated antibodies)

  • Correlate protein detection with mRNA expression (RT-PCR, RNA-seq)

  • Functional validation through inhibition or activation of VEGFR2 signaling

• Technical controls:

  • Secondary antibody-only controls to assess background

  • Isotype controls to evaluate non-specific binding

  • Positive controls using samples with confirmed VEGFR2 expression

This comprehensive validation approach ensures that experimental results accurately reflect VEGFR2 biology rather than technical artifacts.

How should I analyze and quantify VEGFR2 expression in immunohistochemistry experiments?

Quantitative analysis of VEGFR2 expression using KDR (Ab-1214) Antibody in immunohistochemistry requires systematic approaches:

• Staining assessment parameters:

  • Intensity scoring: Establish a consistent scale (0 = negative, 1+ = weak, 2+ = moderate, 3+ = strong)

  • Distribution assessment: Estimate percentage of positive cells (0-100%)

  • Combined scoring: Calculate H-score (Σ[intensity × % positive cells]), ranging from 0-300

  • Subcellular localization: Document membrane, cytoplasmic, and/or nuclear staining patterns

• Quantification methodologies:

  • Manual scoring: At least two independent observers blinded to experimental conditions

  • Digital image analysis: Software-based quantification for more objective assessment

  • Vessel counting: For angiogenesis studies, count VEGFR2-positive vessels per high-power field

• Statistical analysis approaches:

  • Use appropriate statistical tests based on data distribution

  • For normally distributed data: t-tests, ANOVA with post-hoc tests

  • For non-normally distributed data: Mann-Whitney U, Kruskal-Wallis tests

  • Consider correlation analysis with clinical parameters or other biomarkers

• Controls and normalization:

  • Include positive and negative controls in each staining batch

  • Consider normalization to vessel density when appropriate

  • Account for tissue-specific background levels

The images showing immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using VEGFR2(Ab-1214) Antibody demonstrate both the expected staining pattern and the effective use of a blocking peptide control , providing a reference for proper methodology.

What approaches can I use to investigate VEGFR2 signaling mechanisms with this antibody?

Investigating VEGFR2 signaling mechanisms using KDR (Ab-1214) Antibody can be approached through several methodologies:

• Receptor activation studies:

  • Combine KDR (Ab-1214) Antibody with phospho-specific antibodies to assess activation ratio

  • Treatment time courses with VEGF or other stimuli to map activation kinetics

  • Inhibitor studies to identify regulatory mechanisms

• Downstream signaling analysis:

  • Correlate VEGFR2 expression/activation with downstream effectors (ERK, Akt, PLCγ)

  • Use pharmacological inhibitors to define pathway dependencies

  • Design co-immunoprecipitation experiments to identify binding partners

• Spatial organization analysis:

  • Immunofluorescence co-localization studies with cellular compartment markers

  • Investigation of receptor clustering using super-resolution microscopy

  • Analysis of VEGFR2 distribution in specialized membrane domains

• Functional correlation approaches:

  • Link VEGFR2 expression patterns to functional outcomes (proliferation, migration, tube formation)

  • Develop intervention studies (siRNA, inhibitors) to establish causality

  • Use time-lapse imaging with live-cell markers to track dynamics

• Data integration strategies:

  • Correlation analysis between protein data and transcriptomic/proteomic datasets

  • Pathway modeling to place VEGFR2 findings in broader signaling context

  • Multi-parameter analysis to identify regulatory networks

This multi-faceted approach leverages KDR (Ab-1214) Antibody's ability to detect total VEGFR2 as a foundation for comprehensive signaling studies, especially when combined with phospho-specific reagents and functional assays.

How can I use KDR (Ab-1214) Antibody in developmental studies of vascular biology?

KDR (Ab-1214) Antibody can be applied to developmental vascular biology research through these methodological approaches:

• Embryonic vascular development:

  • Whole-mount immunostaining of developing vascular networks

  • Tissue section analysis to track VEGFR2 expression changes throughout development

  • Co-staining with developmental stage markers to create expression timelines

• Methodological considerations:

  • Species reactivity (confirmed for human and mouse ) is critical for model selection

  • Optimization of fixation protocols for embryonic tissues

  • Careful selection of antigen retrieval methods to preserve delicate structures

• Comparative analysis approaches:

  • Assessment of VEGFR2 expression in normal versus pathological development

  • Spatiotemporal mapping of expression in different vascular beds

  • Correlation with functional vascular parameters (perfusion, permeability)

• Intervention studies:

  • Effects of genetic models (conditional knockouts, reporter lines)

  • Response to VEGF pathway modulators during development

  • Recovery dynamics following developmental disruptions

• Analytical frameworks:

  • 3D reconstruction of vascular networks with associated VEGFR2 expression

  • Quantitative assessment of vascular density correlated with VEGFR2 levels

  • Cell-type specific expression patterns across developmental timepoints

This application leverages the antibody's validated reactivity in both human and mouse systems, though optimization for specific developmental stages would be necessary.

What are the considerations for using KDR (Ab-1214) Antibody in conjunction with other VEGFR family receptor antibodies?

When designing multi-receptor studies incorporating KDR (Ab-1214) Antibody alongside antibodies against other VEGFR family members:

• Technical compatibility considerations:

  • Host species compatibility for multiple immunostaining

  • Optimization of common protocols that maintain epitope integrity for all targets

  • Selection of non-overlapping detection systems for simultaneous visualization

• Experimental design strategies:

  • Serial sections for IHC when using antibodies from the same host species

  • Sequential staining protocols with appropriate blocking between steps

  • Controls to assess potential cross-reactivity between antibodies

• Data integration approaches:

  • Correlation analysis between different VEGFR family members

  • Ratio calculations (e.g., VEGFR2:VEGFR1) to assess receptor balance

  • Spatial relationship mapping in complex tissues

• Functional interpretation frameworks:

  • Comparative response to ligands that bind multiple receptors

  • Differential activation of downstream pathways

  • Compensatory regulation between family members

• Validation requirements:

  • Confirmation of antibody specificity for each VEGFR family member

  • Testing for potential cross-reactivity between related epitopes

  • Verification that detection of one receptor doesn't interfere with another

This multi-receptor approach provides a more comprehensive understanding of VEGF signaling networks than single-receptor studies, particularly for complex processes like angiogenesis where receptor balance is critical.

What are common issues with Western blot detection using KDR (Ab-1214) Antibody and how can they be resolved?

When using KDR (Ab-1214) Antibody for Western blot applications, researchers may encounter several technical challenges:

• Weak or absent signal:

  • Increase antibody concentration (recommended range: 1:500-1:3000 )

  • Optimize protein loading (VEGFR2 is ~230 kDa; consider using 50-100 µg total protein)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use more sensitive detection systems (enhanced chemiluminescence substrates)

  • Verify protein transfer efficiency for high molecular weight proteins

• High background:

  • Increase blocking time and concentration (5% BSA or milk)

  • Extend wash steps (at least 3x10 minutes with TBST)

  • Dilute antibody in fresh blocking buffer

  • Reduce secondary antibody concentration

  • Use high-quality, fresh membranes

• Multiple bands or unexpected band size:

  • Include positive control (A431 cells show a specific band at ~230 kDa )

  • Use freshly prepared samples with protease inhibitors

  • Optimize sample preparation to prevent degradation of high molecular weight proteins

  • Consider the possibility of detecting VEGFR2 isoforms or post-translational modifications

• Inconsistent results:

  • Standardize lysate preparation protocols

  • Use consistent antibody lot numbers when possible

  • Maintain consistent exposure times for quantitative comparisons

  • Include internal controls in each experiment

Following the demonstrated protocol that successfully detected VEGFR2 in A431 human epithelial carcinoma cell line provides a validated starting point for optimization.

How can I optimize immunofluorescence protocols with KDR (Ab-1214) Antibody?

Optimizing immunofluorescence protocols with KDR (Ab-1214) Antibody requires attention to multiple parameters:

• Sample preparation optimization:

  • Test different fixation methods (the antibody works with methanol-fixed MCF cells )

  • Optimize permeabilization conditions for accessing intracellular epitopes

  • Consider antigen retrieval methods if signal is weak

• Antibody dilution optimization:

  • Start with recommended dilution range (1:100-1:200 )

  • Perform titration experiments to determine optimal concentration

  • Extend primary antibody incubation time (overnight at 4°C) for weak signals

• Signal enhancement strategies:

  • Use tyramide signal amplification for low abundance targets

  • Select appropriate fluorophore-conjugated secondary antibodies

  • Optimize microscope settings for detection sensitivity

• Background reduction approaches:

  • Extend blocking time (1-2 hours with 5-10% normal serum)

  • Include protein blockers (BSA) and detergents (0.1-0.3% Triton X-100)

  • Add extra wash steps with agitation

  • Include autofluorescence quenching steps if needed

• Controls and validation:

  • Include secondary-only controls to assess background

  • Use blocking peptide controls to confirm specificity

  • Compare staining pattern with published literature

The immunofluorescence staining of methanol-fixed MCF cells shown in the search results demonstrates successful application of the antibody and provides a reference pattern for proper optimization.

How can KDR (Ab-1214) Antibody be used in research involving VEGFR2-targeted therapeutics?

KDR (Ab-1214) Antibody can support VEGFR2-targeted therapeutic research through several methodological approaches:

• Target validation strategies:

  • Confirm VEGFR2 expression in target tissues or disease models

  • Quantify receptor levels to predict potential therapeutic response

  • Evaluate heterogeneity of expression across patient samples or model systems

• Mechanism-of-action studies:

  • Monitor changes in total VEGFR2 levels following treatment

  • Combine with phospho-specific antibodies to assess inhibition of activation

  • Track receptor internalization or degradation in response to therapeutic agents

• Resistance mechanism investigation:

  • Analyze changes in VEGFR2 expression in resistant models

  • Detect altered receptor localization or post-translational modifications

  • Identify compensatory pathway activation in treatment-resistant contexts

• Combination therapy evaluation:

  • Assess VEGFR2 expression/activation when combining multiple targeted agents

  • Monitor receptor status during sequential therapy approaches

  • Correlate VEGFR2 dynamics with treatment efficacy markers

• Biomarker development:

  • Standardize VEGFR2 detection protocols for potential diagnostic applications

  • Correlate baseline VEGFR2 expression with treatment response

  • Develop quantitative IHC scoring systems for potential clinical application

This application leverages the antibody's ability to detect total VEGFR2 levels regardless of activation state, providing critical information about target availability and response to therapeutic intervention.

What are the considerations for using KDR (Ab-1214) Antibody in single-cell analysis techniques?

Adapting KDR (Ab-1214) Antibody for single-cell analysis techniques requires special considerations:

• Flow cytometry optimization:

  • Cell fixation and permeabilization for intracellular VEGFR2 detection

  • Titration of antibody concentration for optimal signal-to-noise ratio

  • Selection of appropriate fluorophore-conjugated secondary antibodies

  • Inclusion of viability dyes to exclude dead cells

• Mass cytometry (CyTOF) adaptation:

  • Metal-conjugated secondary antibodies for detection

  • Optimization of signal amplification for lower abundance targets

  • Integration into multi-parameter panels with other signaling markers

  • Careful titration to minimize signal spillover

• Single-cell imaging approaches:

  • Optimization for high-content imaging platforms

  • Balance between signal strength and photobleaching

  • Co-staining strategies for cellular compartment identification

  • Automated image analysis algorithm development

• scRNA-seq integration:

  • Protein-level validation of transcriptomic findings

  • Development of protocols for simultaneous protein and RNA detection

  • Correlation between VEGFR2 protein levels and mRNA expression

• Technical validation requirements:

  • Sensitivity assessment using cells with known VEGFR2 expression levels

  • Specificity confirmation through blocking experiments

  • Benchmarking against established VEGFR2 detection methods