Phospho-PTK2 (S843) Antibody

What is PTK2 and what is the significance of its phosphorylation at serine 843?

PTK2, also known as Focal Adhesion Kinase 1 (FAK), FRNK, FADK1, or p125FAK, is a non-receptor protein tyrosine kinase that plays crucial roles in cell adhesion, migration, and proliferation through its tyrosine kinase activity. It's ubiquitously expressed across various tissues and positively regulates cell population growth, ubiquitin-dependent protein degradation, and protein phosphorylation processes .

Phosphorylation at serine 843 represents a specific post-translational modification that regulates PTK2 activity. Unlike the more commonly studied tyrosine phosphorylation sites (such as Y397), S843 phosphorylation provides a distinct regulatory mechanism that can influence PTK2's interaction with other signaling molecules and its downstream effects on cellular processes. This phosphorylation site is particularly important in understanding the complex regulation of focal adhesion dynamics and cellular migration pathways.

What species reactivity can I expect from commercially available Phospho-PTK2 (S843) antibodies?

Most commercially available Phospho-PTK2 (S843) antibodies demonstrate cross-reactivity with Human, Mouse, and Rat samples. This cross-species reactivity makes these antibodies versatile tools for comparative studies across different model organisms . When planning experiments involving other species, it's advisable to perform preliminary validation tests or consult manufacturer specifications for confirmed reactivity profiles.

What are the recommended applications for Phospho-PTK2 (S843) antibodies?

Phospho-PTK2 (S843) antibodies are primarily validated for Western Blot (WB) applications, making them suitable for detecting and quantifying phosphorylated PTK2 in cell and tissue lysates . While Western blotting represents the most validated application, researchers should consider the following application-specific considerations:

How should I store and handle Phospho-PTK2 (S843) antibodies for optimal performance?

For long-term storage, maintain Phospho-PTK2 (S843) antibodies at -20°C for up to one year. For frequent use and short-term storage (up to one month), store at 4°C to avoid repeated freeze-thaw cycles that may compromise antibody integrity and performance . The antibodies are typically formulated in PBS with 0.02% sodium azide and 50% glycerol at pH 7.2, which helps maintain stability during storage.

When handling the antibody:

• Aliquot upon first thaw to minimize freeze-thaw cycles

• Thaw completely before use and mix gently to ensure homogeneity

• Avoid contamination by using sterile technique

• Return to appropriate storage conditions immediately after use

How does phosphorylation at S843 functionally differ from other PTK2 phosphorylation sites, particularly Y397?

The phosphorylation of PTK2 at different residues creates a complex regulatory network with distinct functional outcomes. S843 phosphorylation represents a serine/threonine kinase-mediated regulation that differs fundamentally from the tyrosine phosphorylation at Y397, which is primarily associated with autophosphorylation and kinase activation.

Comparative analysis of S843 versus Y397 phosphorylation:

Understanding these differential phosphorylation patterns is critical for accurately interpreting experimental results when using phospho-specific antibodies in research contexts involving cell migration, adhesion, and proliferation studies.

What are the most effective positive and negative controls for validating Phospho-PTK2 (S843) antibody specificity in experimental settings?

Rigorous validation of antibody specificity is essential for reliable research outcomes. For Phospho-PTK2 (S843) antibodies, consider the following controls:

Positive Controls:

• Cell lines treated with agents known to induce S843 phosphorylation (e.g., certain growth factors or serum stimulation)

• Recombinant phosphorylated PTK2 protein (if available)

• Cell lines with constitutively high PTK2 activity (certain cancer cell lines)

Negative Controls:

• PTK2 knockout/knockdown cells or tissues

• Samples treated with phosphatase to remove phosphorylation

• Competition with phospho-peptide corresponding to the S843 region

• Mutant cell lines where S843 is replaced with alanine (S843A)

Validation Approach:
Implement a multi-step validation protocol including:

• Peptide competition assays to confirm epitope specificity

• Phosphatase treatment of samples to confirm phospho-specificity

• Sibling antibody comparison (compare with antibodies recognizing total PTK2)

• Cross-validation with alternative detection methods (e.g., mass spectrometry)

How can I distinguish between S843 phosphorylation signals and potential cross-reactivity with other phosphorylation sites in PTK2?

Cross-reactivity represents a significant challenge when working with phospho-specific antibodies. To distinguish true S843 phosphorylation from potential cross-reactivity:

• Phospho-site mutant analysis: Generate S843A mutants where the serine is replaced with alanine, preventing phosphorylation at this specific site.

• Phospho-peptide arrays: Test antibody against peptide arrays containing various PTK2 phosphorylation sites to identify potential cross-reactivity.

• Comparative phosphatase treatment: Use different phosphatases with varying specificities to differentially remove phosphorylation at serine versus tyrosine residues.

• Sequential immunoprecipitation: Perform immunoprecipitation with antibodies against different phosphorylation sites to isolate specific phospho-forms.

• High-resolution techniques: Consider confirmatory analysis with mass spectrometry or Phos-tag gel electrophoresis to definitively identify phosphorylation sites.

What is the relationship between PTK2 S843 phosphorylation and its role in specific cellular signaling pathways or disease states?

PTK2 S843 phosphorylation has been implicated in the regulation of several cellular processes and disease mechanisms:

The connection between S843 phosphorylation and these pathways suggests potential applications in both basic research and translational medicine, particularly in contexts where PTK2 dysfunction is associated with pathological conditions such as leiomyomas, small cell lung cancers, and pulmonary hypertension .

What are the optimal sample preparation protocols for detecting PTK2 S843 phosphorylation in different experimental systems?

Sample preparation critically affects phosphorylation detection. Consider these system-specific protocols:

For Cell Culture Systems:

• Rapid harvesting to preserve phosphorylation status

• Lysis buffer composition: 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS

• Critical additives:

  • Phosphatase inhibitors (10mM sodium fluoride, 1mM sodium orthovanadate)

  • Protease inhibitors (1mM PMSF, protease inhibitor cocktail)

• Maintain samples at 4°C throughout processing

• Sonication (3 × 10s pulses) to ensure complete lysis

For Tissue Samples:

• Flash-freezing immediately after collection

• Homogenization in specialized buffer containing higher detergent concentrations

• Extended extraction time (30-60 minutes) with gentle agitation

• Additional centrifugation steps to remove debris

What are the recommended troubleshooting approaches for common issues when using Phospho-PTK2 (S843) antibodies?

How can I effectively implement Phospho-PTK2 (S843) antibodies in multiplex immunofluorescence or co-immunoprecipitation studies?

For Multiplex Immunofluorescence:

• Antibody selection: Choose antibodies raised in different host species to allow for distinguishable secondary antibody detection

• Sequential staining protocol:

  • Apply Phospho-PTK2 (S843) antibody first

  • Apply other primary antibodies in order of decreasing sensitivity

  • Include appropriate negative controls for each antibody

• Signal amplification strategies: Consider tyramide signal amplification for low-abundance phospho-epitopes

• Spectral unmixing: Implement computational approaches to separate overlapping fluorescence signals

For Co-Immunoprecipitation Studies:

• Buffer optimization: Use mild lysis conditions (1% NP-40 or 0.5% Triton X-100) to preserve protein-protein interactions

• Pre-clearing strategy: Pre-clear lysates with control IgG to reduce non-specific binding

• Antibody immobilization: Consider covalent coupling to beads to prevent antibody contamination in eluted samples

• Two-step verification:

  • IP with Phospho-PTK2 (S843) antibody, then Western blot for interacting partners

  • IP with antibodies against suspected interacting partners, then Western blot with Phospho-PTK2 (S843) antibody

What analytical techniques can enhance quantification of PTK2 S843 phosphorylation beyond standard Western blotting?

While Western blotting is the most validated application for Phospho-PTK2 (S843) antibodies, several advanced techniques offer enhanced quantification capabilities:

• Quantitative immunofluorescence: Provides spatial information about phosphorylation patterns within cells

  • Implementation: Fixed cell imaging with calibrated fluorescence standards

  • Analysis: Nuclear/cytoplasmic ratio, focal adhesion localization quantification

• Flow cytometry for phospho-proteins:

  • Implementation: Permeabilization with methanol fixation

  • Advantage: Single-cell resolution and high-throughput analysis

• Phospho-protein arrays:

  • Implementation: Reverse-phase protein arrays with multiple samples

  • Advantage: Higher throughput than Western blotting

• Mass spectrometry-based approaches:

  • Implementation: Immunoprecipitation followed by LC-MS/MS

  • Advantage: Absolute quantification and detection of multiple phosphorylation sites simultaneously

• ELISA-based methods:

  • Implementation: Sandwich ELISA with capture and detection antibodies

  • Advantage: Higher sensitivity and throughput than Western blotting

How should I design experiments to study temporal dynamics of PTK2 S843 phosphorylation in response to different stimuli?

Designing experiments to capture temporal phosphorylation dynamics requires careful consideration of multiple factors:

• Time course optimization:

  • Include early time points (30 seconds, 1, 2, 5 minutes)

  • Include intermediate time points (15, 30, 60 minutes)

  • Include late time points (2, 6, 24 hours)

  • Use synchronized cell populations where applicable

• Stimuli considerations:

  • Concentration gradients to determine dose-dependent effects

  • Pulse-chase experiments for reversibility assessment

  • Combination stimuli to analyze pathway crosstalk

• Analysis methods:

  • Quantitative Western blotting with phospho-to-total protein normalization

  • Live-cell imaging with phospho-specific biosensors (if available)

  • Computational modeling of phosphorylation kinetics

• Controls:

  • Vehicle controls at each time point

  • Pathway inhibitor controls to validate specificity

  • Phosphatase treatment controls

What kinase pathways are involved in regulating PTK2 S843 phosphorylation and how can I experimentally manipulate them?

Understanding the kinase pathways that regulate S843 phosphorylation is essential for comprehensive PTK2 signaling studies:

Experimental Validation Approach:

• Pharmacological inhibition coupled with stimulus

• Genetic approaches (kinase knockdown/knockout, overexpression of constitutively active/dominant negative mutants)

• In vitro kinase assays with recombinant proteins

• Correlation analysis of kinase activity and S843 phosphorylation status

What considerations should be made when interpreting PTK2 S843 phosphorylation data in the context of cell adhesion and migration studies?

When studying S843 phosphorylation in cell adhesion and migration contexts, consider these interpretive frameworks:

• Spatial distribution matters:

  • Leading edge versus trailing edge phosphorylation patterns

  • Focal adhesion-localized versus cytoplasmic phosphorylation

  • Relationship to other focal adhesion components (paxillin, vinculin)

• Temporal relationship to adhesion dynamics:

  • Early spreading phase

  • Mature adhesion phase

  • Adhesion disassembly phase

• Cell type-specific variations:

  • Epithelial versus mesenchymal cells

  • Normal versus transformed cells

  • 2D versus 3D culture systems

• Mechanical force considerations:

  • Substrate stiffness effects on phosphorylation

  • Stretch/shear stress response

  • Relationship to mechanosensing pathways

• Integration with other signaling pathways:

  • Coordination with Rho GTPase activity

  • Interplay with integrin activation status

  • Relationship to matrix metalloproteinase activity

How are emerging technologies enhancing the detection and functional analysis of PTK2 S843 phosphorylation?

Recent technological advances are transforming our ability to study phosphorylation events with unprecedented precision:

• CRISPR-based approaches:

  • Endogenous tagging of PTK2 for live imaging

  • Site-specific mutation of S843 to study functional consequences

  • CRISPRa/CRISPRi for controlled expression studies

• Proximity labeling methods:

  • BioID or APEX2 fusions to identify proteins interacting specifically with phosphorylated S843

  • Spatially restricted enzymatic tagging to map compartment-specific interactions

• Advanced microscopy techniques:

  • Super-resolution microscopy for nanoscale localization

  • FRET-based biosensors for real-time phosphorylation monitoring

  • Correlative light and electron microscopy for ultrastructural context

• Single-cell phosphoproteomics:

  • Mass cytometry (CyTOF) with phospho-specific antibodies

  • Microfluidic-based single-cell Western blotting

  • Integration with transcriptomic data for multi-omic analysis

What are the promising research directions for understanding the role of PTK2 S843 phosphorylation in disease pathogenesis and therapeutic development?

The involvement of PTK2 in multiple disease processes suggests several promising research directions:

• Cancer biology applications:

  • Correlation of S843 phosphorylation with invasion and metastasis markers

  • Evaluation as a prognostic biomarker

  • Target for developing selective inhibitors that modulate S843-specific functions

• Cardiovascular disease research:

  • Role in vascular remodeling and angiogenesis

  • Connection to pulmonary hypertension pathophysiology

  • Potential marker for endothelial dysfunction

• Inflammatory disorders:

  • Function in immune cell migration and activation

  • Role in inflammatory signaling cascades

  • Response to inflammatory mediators

• Therapeutic development approaches:

  • Structure-based drug design targeting S843 binding pocket

  • Development of proteolysis targeting chimeras (PROTACs) for selective degradation

  • Gene therapy approaches to modulate PTK2 function in specific tissues