Phospho-KDR (Tyr1214) Antibody

What are the primary research applications for Phospho-KDR (Tyr1214) antibodies?

Phospho-KDR (Tyr1214) antibodies are versatile research tools that can be employed across multiple experimental platforms. These antibodies are specifically validated for Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), and Enzyme-Linked Immunosorbent Assay (ELISA) applications . Western blotting represents the most common application for detecting phosphorylation status at Tyr1214, typically requiring dilutions ranging from 1:500 to 1:2000 depending on the specific antibody formulation and experimental conditions . For immunohistochemical applications, researchers typically utilize dilutions between 1:100 and 1:300 to achieve optimal staining with minimal background . Immunofluorescence applications generally perform best at dilutions between 1:200 and 1:1000, while ELISA applications may require much higher dilutions, often around 1:10000 . Each application requires specific optimization protocols to ensure specificity and sensitivity when detecting this phosphorylation site.

How is specificity for phosphorylated Tyr1214 verified in experimental settings?

Specificity verification for Phospho-KDR (Tyr1214) antibodies involves multiple validation approaches to ensure selective recognition of the phosphorylated form. The primary validation method involves comparative phosphopeptide mapping, where researchers generate tryptic peptides from wild-type and Y1214F mutant KDR/Flk-1 proteins . When analyzing these peptide maps, the Y1214F mutant displays a characteristic absence of a major phosphopeptide spot that is present in wild-type samples, confirming the specificity for this phosphorylation site . Additional validation often includes immunoblotting against phosphorylated versus non-phosphorylated recombinant proteins, peptide competition assays, and immunoprecipitation followed by mass spectrometry analysis. For successful experimental verification, researchers should include appropriate positive controls (VEGF-A stimulated endothelial cells) and negative controls (cells treated with tyrosine kinase inhibitors or phosphatase treatment of lysates) to confirm antibody specificity for the phosphorylated epitope within the amino acid range 1180-1229 .

What are the optimal cell models for studying KDR Tyr1214 phosphorylation?

Several established cell models have demonstrated reliable KDR Tyr1214 phosphorylation responses in research settings. Human umbilical vein endothelial cells (HUVECs) represent the gold standard for studying VEGFR2/KDR signaling and phosphorylation events in primary human endothelial cells . Mouse aortic endothelial cells (MAECs) and mouse venous endothelial cells (MVECs) provide valuable models for studying species-specific differences in KDR signaling . Interestingly, recent research has also validated PC12 neuronal cells as an alternative model for investigating the neuronal aspects of VEGFR2/KDR signaling and phosphorylation . For heterologous expression studies, HEK293 cells have been successfully employed to express wild-type and mutant KDR/Flk-1 constructs for mechanistic studies of receptor phosphorylation . When designing experiments, researchers should select cell models based on their specific research questions, considering the endogenous expression levels of KDR, the presence of co-receptors, and the completeness of downstream signaling pathways in each model system.

What stimulation protocols most effectively induce KDR Tyr1214 phosphorylation?

Effective induction of KDR Tyr1214 phosphorylation requires optimized stimulation protocols tailored to the experimental system. The most robust stimulation is achieved using recombinant VEGF-A at concentrations of 50 ng/μL, which reliably triggers receptor dimerization and subsequent autophosphorylation at multiple tyrosine residues including Tyr1214 . VEGF-B can also be used as a stimulus at similar concentrations, although it typically produces a different phosphorylation profile compared to VEGF-A . The optimal stimulation duration varies by experimental endpoint: for acute signaling studies, 5-15 minute stimulations are generally sufficient to observe maximal Tyr1214 phosphorylation, while longer stimulations (30 minutes to several hours) may be required for studying downstream biological effects such as neurite outgrowth or endothelial cell migration. Prior to stimulation, cells should be serum-starved for 4-6 hours to reduce baseline phosphorylation levels. Stimulation temperature is also critical, with 37°C being optimal for maintaining physiological receptor kinetics. Researchers should establish time-course and dose-response curves in their specific experimental systems to determine optimal conditions.

How can researchers overcome detection challenges when working with tissue samples?

Detection of phosphorylated KDR Tyr1214 in tissue samples presents several technical challenges that require specific methodological refinements. The primary challenge is preserving phosphorylation status during tissue processing, as phosphoepitopes are highly susceptible to degradation by endogenous phosphatases. Researchers should implement a rapid tissue harvesting protocol with immediate fixation in phosphatase inhibitor-supplemented fixatives . For immunohistochemical applications, antigen retrieval optimization is critical - heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) typically yields superior results for exposing the Tyr1214 phosphoepitope . Background signal reduction can be achieved through careful blocking optimization, using 5% BSA with 0.3% Triton X-100 for permeabilization when performing immunofluorescence on tissue sections . For frozen tissue sections, acetone fixation for 10 minutes at -20°C helps preserve phosphoepitopes while maintaining tissue architecture. When working with archival paraffin-embedded samples, researchers should be aware that long-term storage may significantly reduce phosphoepitope detection sensitivity, requiring extended antibody incubation times (overnight at 4°C) and signal amplification techniques such as tyramide signal amplification to visualize phospho-KDR Tyr1214.

What are effective troubleshooting strategies for inconsistent Western blot results?

Inconsistent Western blot results when detecting phospho-KDR (Tyr1214) can stem from multiple factors throughout the experimental workflow. Sample preparation represents a critical determinant of success - lysates must be prepared with robust phosphatase inhibitor cocktails, maintained at cold temperatures throughout processing, and analyzed freshly whenever possible . When technical inconsistencies arise, researchers should systematically evaluate and optimize several parameters: (1) Sample denaturation conditions - complete denaturation in SDS sample buffer at 95°C for 5 minutes typically yields optimal results for exposing the phosphoepitope; (2) Protein loading amount - 30-50 μg of total protein per lane generally provides sufficient KDR detection in most cell types; (3) Transfer conditions - wet transfer at constant amperage (rather than voltage) improves reproducibility for high molecular weight proteins like KDR (152 kDa) ; (4) Blocking conditions - 5% BSA in TBST is superior to milk-based blockers which contain phosphatases that can dephosphorylate antigens; (5) Antibody dilution and incubation time - primary antibody at 1:1000 dilution incubated overnight at 4°C typically produces optimal signal-to-noise ratios . When troubleshooting, always include positive controls (VEGF-stimulated endothelial cells) and loading controls (total KDR, β-actin) to normalize phosphorylation signals and account for lane-to-lane variations.

How should researchers quantitatively analyze Tyr1214 phosphorylation in relation to total KDR levels?

Quantitative analysis of Tyr1214 phosphorylation requires normalization to total KDR protein levels to accurately assess the proportion of phosphorylated receptor. The recommended analytical approach employs dual detection methods where samples are analyzed in parallel for both phosphorylated and total KDR protein . For Western blot analysis, researchers should implement a sequential probing method: first detecting phospho-KDR (Tyr1214), then stripping and reprobing the same membrane for total KDR, followed by digital image capture and densitometric analysis using software such as ImageJ or LI-COR Odyssey systems . The phosphorylation index is then calculated as the ratio of phospho-KDR band intensity to total KDR band intensity, which controls for variations in total receptor expression across samples. For cell-based ELISAs, normalization can be performed using dual-color detection systems where phospho-KDR and total KDR are simultaneously detected with different chromogenic or fluorescent substrates . When presenting phosphorylation data, researchers should report both the absolute values and normalized ratios, along with statistical analyses comparing treatment conditions. This approach ensures that observed changes in phosphorylation status represent genuine biological effects rather than artifacts from variable protein expression or loading.

How does phosphorylation at Tyr1214 compare with other KDR phosphorylation sites in experimental detection?

KDR/VEGFR2 contains multiple tyrosine phosphorylation sites with distinct functional implications, requiring careful comparative analysis approaches. Research has established that Y1175 and Y1214 represent the two major autophosphorylation sites on KDR/Flk-1 both in vitro and in vivo, while other sites like Y801 show comparatively lower phosphorylation levels . When conducting comparative phosphorylation site analysis, researchers should employ phosphosite-specific antibodies with validated specificity for each tyrosine residue . Multiplexed detection approaches, such as multistrip Western blotting or multiplex flow cytometry, allow simultaneous assessment of multiple phosphorylation sites from the same sample. Phosphopeptide mapping has revealed distinctive tryptic peptide patterns for Y1175 versus Y1214 phosphorylation, with Y1214F mutants displaying the absence of a characteristic major spot observed in wild-type KDR/Flk-1 . While Y1175 phosphorylation is strongly associated with PLCγ1 recruitment and MAPK pathway activation, Y1214 phosphorylation appears to regulate distinct signaling pathways not directly linked to MAP kinase activation . This phosphosite-specific signaling divergence underscores the importance of analyzing multiple phosphorylation sites when studying KDR/VEGFR2 biology in different cellular contexts.

What is the functional significance of KDR Tyr1214 phosphorylation in endothelial versus neuronal cells?

The functional significance of KDR Tyr1214 phosphorylation exhibits cell type-specific patterns with distinct biological outcomes in endothelial versus neuronal contexts. In endothelial cells, Tyr1214 phosphorylation contributes to vascular development and angiogenic processes through activation of pathways distinct from the classical MAP kinase cascade . This phosphorylation event appears particularly important for endothelial cell motility and reorganization of the actin cytoskeleton, which are essential components of angiogenic responses . In contrast, recent research has identified novel roles for KDR Tyr1214 phosphorylation in neuronal contexts, particularly in PC12 neuronal cells, where it influences neurite growth and potentially contributes to nerve regeneration mechanisms . The cell type-specific outcomes likely result from differential expression of downstream effector proteins and signaling adapters in endothelial versus neuronal cellular environments. While the VEGF-A-induced tyrosine phosphorylation of KDR leads to similar receptor activation in both cell types, the downstream biological consequences diverge significantly based on the cellular context. This functional divergence highlights the importance of studying KDR Tyr1214 phosphorylation in multiple cell types to fully understand its contextual biology.

How does Tyr1214 phosphorylation contribute to pathological angiogenesis in disease models?

Dysregulated KDR Tyr1214 phosphorylation contributes to pathological angiogenesis through aberrant activation of specific downstream signaling cascades. In cancer models, hyperphosphorylation of KDR at Tyr1214 correlates with increased tumor angiogenesis and more aggressive disease progression . This enhanced phosphorylation promotes excessive endothelial cell migration and formation of disorganized, leaky vasculature characteristic of tumor microenvironments. In models of diabetic retinopathy, persistent KDR Tyr1214 phosphorylation contributes to pathological neovascularization in the retina, leading to vision impairment . The phosphorylation at this site appears particularly relevant for inflammatory angiogenesis, where it helps coordinate endothelial responses to both VEGF and inflammatory cytokines. Therapeutic approaches targeting KDR signaling in pathological contexts include both direct kinase inhibitors and antibodies that prevent VEGF binding to the receptor, thereby reducing Tyr1214 phosphorylation . Experimental disease models utilizing Y1214F mutant receptors have demonstrated reduced pathological angiogenesis while maintaining normal vascular homeostasis, suggesting this phosphorylation site might represent a more selective therapeutic target compared to complete VEGFR2/KDR inhibition. These findings highlight the potential for developing phosphosite-specific therapeutic approaches that selectively target pathological angiogenic processes while preserving normal vascular function.

What downstream signaling pathways are specifically activated by KDR Tyr1214 phosphorylation?

KDR Tyr1214 phosphorylation activates specific downstream signaling networks distinct from those mediated by other phosphorylation sites on the receptor. Unlike Tyr1175 phosphorylation, which primarily activates the PLCγ-PKC-MAPK pathway, Tyr1214 phosphorylation does not significantly impact MAP kinase activation . Research has demonstrated that, contrary to what might be expected from its sequence context, Tyr1214 does not serve as a major binding site for the adaptor protein Grb2, as confirmed by pull-down assays using the Grb2 SH2 domain . Instead, Tyr1214 phosphorylation appears more involved in pathways regulating cytoskeletal reorganization and cell motility, which are essential for processes like endothelial cell migration and tube formation during angiogenesis . The precise molecular interactions initiated by Tyr1214 phosphorylation remain an active area of investigation, with current evidence suggesting roles in activating stress-activated protein kinases and potentially modulating small GTPase function. In neuronal contexts, Tyr1214 phosphorylation contributes to neurite outgrowth signaling pathways that remain mechanistically distinct from classic angiogenic pathways . Further research utilizing phosphosite-specific mutants and interactome analysis is needed to fully elucidate the complete signaling network downstream of this important phosphorylation site.

How can phosphoproteomics approaches enhance the study of KDR Tyr1214 phosphorylation dynamics?

Advanced phosphoproteomics methodologies offer powerful approaches for studying KDR Tyr1214 phosphorylation in complex biological systems. Mass spectrometry-based phosphopeptide enrichment techniques, including titanium dioxide (TiO₂) chromatography and immobilized metal affinity chromatography (IMAC), enable unbiased detection of phosphorylation events across the proteome . When applying these approaches to KDR Tyr1214 research, investigators can implement Stable Isotope Labeling with Amino acids in Cell culture (SILAC) or Tandem Mass Tag (TMT) labeling to perform quantitative comparisons across multiple experimental conditions or time points following VEGF stimulation. Phosphoproteomics analysis has the distinct advantage of simultaneously monitoring multiple phosphorylation sites on KDR/VEGFR2, including Tyr1214, Y1175, and other less characterized sites, providing a comprehensive view of receptor activation dynamics . For targeted analysis of Tyr1214 phosphorylation, parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM) mass spectrometry methods can be developed to quantify specific phosphopeptides containing this residue with high sensitivity and specificity. Integration of phosphoproteomics data with computational pathway analysis tools allows researchers to place Tyr1214 phosphorylation in broader signaling networks and predict functional outcomes in different cellular contexts. These advanced methodologies complement traditional antibody-based approaches and provide deeper insights into the temporal and contextual dynamics of KDR phosphorylation events.

What are the latest imaging techniques for visualizing KDR Tyr1214 phosphorylation in live cells and tissues?

Cutting-edge imaging techniques have revolutionized the visualization of KDR Tyr1214 phosphorylation dynamics in biological systems. Förster Resonance Energy Transfer (FRET)-based biosensors represent a powerful approach for monitoring this phosphorylation event in live cells with high spatiotemporal resolution . These biosensors typically incorporate a phospho-specific binding domain (such as a modified SH2 domain) that recognizes the phosphorylated Tyr1214 residue, flanked by fluorescent protein pairs that undergo FRET when the sensor adopts its phosphorylation-dependent conformation. For tissue-level imaging, clearing techniques such as CLARITY, CUBIC, or iDISCO combined with phospho-specific antibodies enable three-dimensional visualization of KDR phosphorylation patterns throughout intact tissues . Super-resolution microscopy methods, including Stimulated Emission Depletion (STED) and Stochastic Optical Reconstruction Microscopy (STORM), provide nanoscale resolution of phosphorylated KDR localization within cellular compartments, revealing previously undetectable spatial organization . For in vivo imaging applications, near-infrared fluorescent (NIRF) labeled antibodies against phospho-KDR (Tyr1214) can be utilized for non-invasive detection of angiogenic activity in disease models. These advanced imaging approaches complement biochemical analyses and provide unique insights into the spatial and temporal dynamics of KDR Tyr1214 phosphorylation in complex biological contexts.

How can monitoring KDR Tyr1214 phosphorylation inform the development of anti-angiogenic therapies?

Monitoring KDR Tyr1214 phosphorylation status provides critical insights for anti-angiogenic therapeutic development and patient stratification strategies. Unlike complete VEGFR2/KDR inhibition, which can cause significant on-target toxicities including hypertension and proteinuria, selective targeting of specific phosphorylation-dependent pathways may offer improved therapeutic windows . Preclinical models have demonstrated that Y1214F mutant forms of KDR/VEGFR2 disrupt pathological angiogenesis while preserving normal vascular homeostasis, suggesting phosphosite-specific inhibition strategies may reduce adverse effects . In drug development pipelines, high-throughput cell-based ELISAs for phospho-KDR (Tyr1214) enable efficient screening of compound libraries to identify molecules that selectively modulate this phosphorylation event . For clinical applications, immunohistochemical assessment of Tyr1214 phosphorylation in tumor biopsies may serve as a biomarker for predicting response to anti-angiogenic therapies, allowing for personalized treatment approaches. Quantitative analysis of phospho-KDR (Tyr1214) levels in circulating endothelial cells or tumor-derived extracellular vesicles could provide non-invasive monitoring of treatment efficacy. Furthermore, understanding the distinct downstream signaling pathways activated by Tyr1214 phosphorylation opens avenues for developing combination therapies that simultaneously target multiple nodes in angiogenic signaling networks, potentially overcoming resistance mechanisms observed with current anti-angiogenic approaches.

What is the potential for developing phosphosite-specific inhibitors targeting KDR Tyr1214?

The development of phosphosite-specific inhibitors targeting KDR Tyr1214 represents an emerging frontier in targeted anti-angiogenic therapy with several promising strategic approaches. Unlike conventional tyrosine kinase inhibitors that block all VEGFR2/KDR signaling, phosphosite-specific approaches aim to selectively disrupt specific downstream pathways while preserving others, potentially improving therapeutic indices . Structure-based drug design strategies utilizing crystal structures of the KDR kinase domain can identify compounds that preferentially inhibit phosphorylation at Tyr1214 through allosteric mechanisms that alter local conformation around this residue. Another promising approach involves developing stabilized peptidomimetics that compete with downstream effector proteins for binding to the phosphorylated Tyr1214 residue, thereby selectively blocking this signaling node. Therapeutic antibodies represent a third strategy, with the potential to develop antibodies that specifically recognize the phosphorylated Tyr1214 epitope and prevent recruitment of downstream signaling molecules . Recent advances in proteolysis-targeting chimeras (PROTACs) technology offer the possibility of developing degraders that selectively target KDR when phosphorylated at specific residues like Tyr1214. While these approaches remain technically challenging, they hold significant promise for developing next-generation anti-angiogenic therapies with improved selectivity profiles compared to current broad-spectrum VEGFR inhibitors, potentially addressing the substantial side effect burden that limits the clinical utility of existing agents.

What storage and handling protocols maximize antibody performance for phospho-KDR (Tyr1214) detection?

Optimal storage and handling protocols are critical for maintaining antibody performance when detecting the phospho-KDR (Tyr1214) epitope. Commercially available antibodies are typically formulated in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide, which stabilizes the immunoglobulin structure and prevents microbial growth . These reagents should be stored at -20°C for up to one year from receipt, with aliquoting into single-use volumes strongly recommended to avoid repeated freeze-thaw cycles that significantly compromise antibody functionality . When preparing working dilutions, researchers should use fresh buffers containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride, and β-glycerophosphate) to prevent dephosphorylation of standards and samples. For immunohistochemical applications, immediate fixation of tissues is essential, as phosphoepitopes are highly labile and rapidly lost during delayed processing . During Western blotting procedures, maintaining cold temperatures throughout sample preparation and adding phosphatase inhibitor cocktails to lysis buffers are critical steps for preserving phosphorylation status . For long-term storage of diluted antibody solutions, addition of carrier proteins (0.5-1% BSA) and antimicrobial agents like sodium azide (0.02%) helps maintain stability. Researchers should perform regular validation of stored antibodies using positive control lysates from VEGF-stimulated endothelial cells to confirm retention of phospho-specificity over time.

What positive and negative controls should be included when validating phospho-KDR (Tyr1214) antibodies?

Rigorous experimental design for phospho-KDR (Tyr1214) detection requires carefully selected positive and negative controls to ensure valid and interpretable results. For positive controls, endothelial cells (HUVECs, MAECs, or MVECs) stimulated with VEGF-A (50 ng/μL) for 5-10 minutes provide reliable Tyr1214 phosphorylation that can be detected across multiple platforms . Cell lines stably transfected with constitutively active VEGFR2/KDR constructs also serve as excellent positive controls with consistent phosphorylation levels. Negative controls should include multiple approaches: (1) Unstimulated cells maintained in serum-free conditions to establish baseline phosphorylation levels; (2) VEGF-stimulated cells pretreated with VEGFR tyrosine kinase inhibitors (e.g., SU5416 or axitinib) to block receptor activation; (3) Cell lysates treated with lambda phosphatase to enzymatically remove phosphate groups; and (4) Ideally, cells expressing the Y1214F mutant form of KDR/VEGFR2, which provides the most specific negative control for this phosphorylation site . For antibody validation, peptide competition assays using the phosphorylated peptide immunogen effectively demonstrate binding specificity. Additionally, siRNA knockdown of KDR/VEGFR2 should eliminate the phospho-signal in Western blots and immunostaining, confirming signal specificity . Implementing this comprehensive panel of controls enables confident interpretation of experimental results and validates the phospho-specificity of the antibody preparation being utilized.

How can researchers quantitatively validate the specificity of phospho-KDR (Tyr1214) antibodies?

Quantitative validation of phospho-KDR (Tyr1214) antibody specificity requires multifaceted approaches that assess both technical performance parameters and biological relevance. Peptide dot blot analysis provides an initial quantitative assessment by comparing antibody binding to phosphorylated versus non-phosphorylated Tyr1214 peptides across a concentration gradient, with specificity indicated by >100-fold higher affinity for the phosphopeptide . For cell-based validation, researchers should implement quantitative Western blotting with recombinant standards of known concentration to establish a linear detection range for the antibody . Specificity can be quantitatively demonstrated through densitometric analysis comparing signal intensity ratios between VEGF-stimulated samples and various negative controls (tyrosine kinase inhibitor treatment, Y1214F mutant expression, phosphatase treatment) . Flow cytometry provides another quantitative platform, where phospho-KDR (Tyr1214) staining intensity can be measured at the single-cell level and expressed as fold-change in mean fluorescence intensity upon VEGF stimulation. For immunohistochemical applications, digital pathology approaches using automated image analysis algorithms can quantify staining intensity across tissue sections, with specificity confirmed by analyzing serial sections stained with primary antibody pre-absorbed with phosphopeptide versus non-phosphopeptide . Finally, mass spectrometry-based validation using immunoprecipitation followed by LC-MS/MS analysis provides the most definitive assessment, confirming that the antibody specifically enriches for peptides containing phosphorylated Tyr1214 of KDR/VEGFR2.