PTK2 Antibody Pair

What is PTK2 and why is its interaction with SRC significant in research?

PTK2 (Protein Tyrosine Kinase 2) is a non-receptor tyrosine kinase also known as FAK, FAK1, or FADK that plays critical roles in cell migration, adhesion, and signaling. The interaction between PTK2 and SRC is particularly significant as these proteins form complexes that regulate multiple downstream pathways involved in cellular migration, invasion, and survival mechanisms. This interaction is detectable using specialized antibody pairs designed for proximity ligation assays (PLA), where each red dot visible in immunofluorescence represents a protein-protein interaction complex between PTK2 and SRC. The biological significance of this interaction extends to multiple research areas including cancer biology, cell motility studies, and signal transduction mechanisms .

In which cellular compartments is PTK2 typically expressed and detected?

PTK2 is predominantly expressed in focal adhesions and cell junctions, making it a critical component of cell-matrix and cell-cell contact points. Researchers have reported positive staining in corpus callosum cell junctions using anti-PTK2 antibodies . When designing experiments to detect PTK2, it's important to consider its subcellular localization primarily in:

• Focal adhesions

• Cell-cell junctions

• Cytoplasmic regions proximal to membrane attachment sites

• Occasionally nuclear localization in certain cell types

Immunofluorescence microscopy with appropriate controls is recommended for accurate subcellular localization studies, as PTK2 distribution may vary depending on cell type and activation state .

What are the optimal techniques for detecting PTK2-protein interactions?

The proximity ligation assay (PLA) is the gold standard technique for detecting PTK2 protein-protein interactions in situ. This method offers several advantages over traditional co-immunoprecipitation:

• Allows visualization of interactions within intact cellular contexts

• Provides spatial information about where interactions occur

• Enables quantification of interaction events at the single-cell level

For optimal results when studying PTK2-SRC interactions using PLA:

• Use anti-PTK2 rabbit polyclonal antibody at 1:1200 dilution

• Use anti-SRC mouse monoclonal antibody at 1:50 dilution

• Each red dot in the resulting images represents a single protein-protein interaction complex

• Analyze images using specialized software such as BlobFinder (available from The Centre for Image Analysis at Uppsala University)

Co-immunoprecipitation remains valuable for biochemical confirmation of interactions, particularly when combined with western blotting to verify protein identity.

How can researchers optimize experimental conditions for PTK2 western blotting?

Successful western blotting for PTK2 requires careful optimization of several experimental parameters. Based on validated protocols, researchers should consider:

Recommended Western Blotting Protocol for PTK2 Detection:

• Sample preparation: Load 30 μg of protein per lane under reducing conditions

• Gel electrophoresis: Use 5-20% gradient SDS-PAGE at 70V (stacking)/90V (resolving) for 2-3 hours

• Transfer conditions: Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

• Blocking: 5% non-fat milk in TBS for 1.5 hours at room temperature

• Primary antibody: Anti-PTK2 antibody at 0.5 μg/mL, incubated overnight at 4°C

• Washing: TBS with 0.1% Tween, 3 times for 5 minutes each

• Secondary antibody: Goat anti-rabbit IgG-HRP at 1:5000 dilution for 1.5 hours at room temperature

• Detection: Enhanced chemiluminescence detection system

• Expected result: PTK2 appears as a specific band at approximately 125 kDa (theoretical size is 119 kDa)

This protocol has been validated across multiple cell lines including HeLa, PC-3, K562, A431, A549, HepG2, rat brain tissue, rat C6, mouse lung tissue, mouse brain tissue, RAW264.7, and NIH/3T3 .

What is the relationship between PTK2 and PTK2B in cellular signaling pathways?

While PTK2 and PTK2B (also known as PYK2) share structural similarities, they function differently in cellular signaling networks:

Key Differences Between PTK2 and PTK2B:

PTK2B directly phosphorylates TBK1 at residue Y591, which increases TBK1 oligomerization and activation. Additionally, PTK2B interacts with STING to promote its oligomerization through a kinase-independent mechanism. These molecular events are critical for mounting effective antiviral innate immune responses .

How can researchers validate PTK2 antibody specificity and minimize non-specific binding?

Validating antibody specificity is crucial for generating reliable research data. For PTK2 antibodies, consider these comprehensive validation approaches:

• Knockout/knockdown controls:

  • Generate Ptk2-knockout cells using CRISPR-Cas9 system

  • Use siRNA knockdown as an alternative approach

  • Verify absence of signal in knockout samples compared to wild-type

• Cross-reactivity assessment:

  • Test antibodies against recombinant PTK2 and structurally similar proteins (especially PTK2B)

  • Compare staining patterns across multiple tissues known to express or lack PTK2

  • Consider potential cross-reactivity with PTK2 isoforms

• Signal verification techniques:

  • Use blocking peptides to confirm signal specificity

  • Employ multiple antibodies targeting different PTK2 epitopes

  • Compare commercial antibodies from different vendors

• Optimized protocols to minimize non-specific binding:

  • For immunofluorescence: Extend blocking time to 2 hours and use 5% BSA in PBS

  • For Western blot: Include 0.1% SDS in wash buffer to reduce background

  • For immunoprecipitation: Pre-clear lysates with protein A/G beads before adding the antibody

What are the common challenges when detecting PTK2 in T-cells and how can they be addressed?

Detecting PTK2 in T-cells presents unique challenges compared to other cell types like fibroblasts or epithelial cells. According to research data and user inquiries:

Common Challenges and Solutions:

• Lower expression levels:

  • Increase protein loading to 40-50 μg per lane for Western blot

  • Extend primary antibody incubation time to 24-36 hours at 4°C

  • Consider protein enrichment techniques like immunoprecipitation before detection

• High background issues:

  • Increase blocking time to 2-3 hours

  • Use 5% BSA instead of milk for blocking

  • Add 0.2% Triton X-100 to permeabilization buffer for immunofluorescence

• Activation-dependent detection:

  • Consider stimulating T-cells (e.g., with anti-CD3/CD28) to increase PTK2 phosphorylation

  • Compare resting vs. activated T-cells

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation status

• Validation approaches:

  • Use T-cell-specific positive controls (literature indicates PTK2 is expressed in T-cells)

  • Include at least one non-T-cell sample as comparison

  • Consider subcellular fractionation to enrich for membrane/cytoskeletal fractions

How does PTK2 inhibition affect resistance mechanisms in cancer therapeutics?

Research indicates PTK2 may play a significant role in therapeutic resistance mechanisms, particularly in the context of EGFR-TKI resistance in non-small cell lung cancer (NSCLC). Experimental evidence suggests:

• Combined targeting approach:

  • Co-inhibition of EGFR and PTK2 may overcome resistance to EGFR-TKI therapy

  • Oral administration of defactinib (PTK2 inhibitor) with osimertinib has been evaluated in mouse xenograft models

• Mechanistic considerations:

  • PTK2 may activate alternative survival pathways when primary oncogenic drivers are inhibited

  • Phosphorylation antibody array assays in PC-9 and PC-9/PEM cell lines have been used to identify PTK2 activation in resistant cells

• Experimental design for studying PTK2 in resistance:

  • Establish resistant cell lines through chronic exposure to targeted therapies

  • Measure cell viability using WST-8 assay when combining PTK2 inhibitors with primary therapies

  • Monitor changes in phosphorylation status of downstream signaling molecules

  • Consider in vivo validation using xenograft models to confirm in vitro findings

What considerations are important when designing experiments to study PTK2-SRC interactions?

When designing experiments to investigate PTK2-SRC interactions, researchers should consider:

• Choice of detection method:

  • Proximity Ligation Assay (PLA) provides in situ visualization of interactions

  • Co-immunoprecipitation confirms biochemical association

  • FRET/BRET approaches can monitor interactions in live cells

• Antibody selection criteria:

  • Use antibodies from different species (e.g., rabbit anti-PTK2 and mouse anti-SRC)

  • Ensure antibodies target regions that don't interfere with the interaction interface

  • Validate antibodies in cells with known PTK2-SRC interaction status

• Experimental controls:

  • Positive control: cells with known PTK2-SRC interaction (e.g., HeLa cells)

  • Negative control: cells treated with SRC inhibitors or following SRC knockdown

  • Technical control: samples where either primary antibody is omitted

• Stimulation conditions:

  • Consider testing both basal and stimulated conditions

  • Cell adhesion to extracellular matrix proteins can enhance PTK2-SRC interactions

  • Growth factor stimulation (e.g., EGF, PDGF) can modulate the interaction

What approaches can be used to study PTK2 domain-specific functions and interactions?

Understanding PTK2 domain-specific functions requires specialized experimental approaches:

Domain Structure of PTK2 and Experimental Approaches:

Recommended approaches for domain-specific studies:

• Domain mapping experiments:

  • Generate truncation constructs expressing specific PTK2 domains

  • Test interaction with putative partners using co-immunoprecipitation

  • Perform domain swapping between PTK2 and PTK2B to identify specificity determinants

• Functional rescue experiments:

  • Knockdown endogenous PTK2 using siRNA targeting UTRs

  • Re-express domain mutants to identify which domains are required for specific functions

  • Monitor cellular phenotypes like migration, adhesion, or signaling

• Advanced imaging approaches:

  • FRET biosensors to monitor domain-specific conformational changes

  • Domain-specific antibodies for immunofluorescence

  • Live-cell imaging with domain-tagged fluorescent proteins

How can PTK2 antibodies be modified for specialized research applications?

Researchers often need to modify antibodies for specialized applications. For PTK2 antibodies:

• Biotin conjugation protocol:

  • Start with antibodies free of BSA and sodium azide

  • Perform buffer exchange to remove preservatives (use PBS)

  • Use NHS-biotin with a 20:1 molar ratio of biotin:antibody

  • Incubate for 2 hours at room temperature

  • Purify using desalting columns

  • Store conjugated antibody at -20°C in small aliquots to avoid freeze-thaw cycles

• Fluorophore conjugation considerations:

  • Choose fluorophores with appropriate spectral properties for your imaging system

  • Consider antibody:fluorophore ratio to prevent over-labeling

  • Maintain antibody concentration above 1 mg/ml during conjugation

  • Test multiple conjugation ratios to optimize signal-to-noise

• Enzyme conjugation for PTK2 detection:

  • HRP or AP conjugation for enhanced sensitivity in immunohistochemistry

  • Maintain enzymatic activity by avoiding harsh conjugation conditions

  • Test conjugated antibodies against native versions to ensure epitope recognition is preserved

What are the latest advances in multiplex detection systems for studying PTK2 signaling networks?

Recent advances in multiplex detection allow simultaneous analysis of PTK2 and its signaling partners:

• Phosphorylation antibody arrays:

  • Enable simultaneous detection of multiple phosphorylated RTKs

  • Have been successfully employed to study PTK2 activation in contexts like EGFR-TKI resistance

  • Allow for comparison between sensitive and resistant cell lines

  • Provide a systems-level view of compensatory signaling mechanisms

• Mass cytometry (CyTOF) approaches:

  • Metal-tagged antibodies enable simultaneous detection of >40 proteins

  • Can be combined with single-cell analysis to identify subpopulations

  • Requires careful antibody panel design and validation

  • Particularly valuable for heterogeneous samples like tumor tissues

• Multiplex immunofluorescence techniques:

  • Sequential staining with tyramide signal amplification

  • Spectral unmixing to resolve overlapping fluorophores

  • Cyclic immunofluorescence for 20+ targets on the same sample

  • Particularly useful for spatial analysis of PTK2 interactions in tissue sections

How is PTK2 research contributing to understanding antiviral immune responses?

Recent findings highlight an emerging role for the PTK2 family in antiviral immunity, particularly through PTK2B:

• PTK2B role in antiviral signaling:

  • PTK2B directly phosphorylates TBK1 at Y591, enhancing TBK1 oligomerization and activation

  • PTK2B promotes STING oligomerization through a kinase-independent mechanism

  • Ptk2b-knockout mice show increased susceptibility to viral infections including HSV-1 and VSV

  • PTK2B depletion reduces antiviral signaling in multiple cell types including MEFs, macrophages and dendritic cells

• Experimental approaches to study PTK2/PTK2B in antiviral responses:

  • Employ CRISPR-Cas9 to generate knockout cells or animals

  • Measure mRNA levels of interferon-stimulated genes (Ifnb1, Ifit1, Cxcl10) using qPCR

  • Assess phosphorylation status of TBK1 and IRF3 following viral challenge

  • Use domain mapping to identify critical regions for protein-protein interactions

• Differences between PTK2 and PTK2B in immune regulation:

  • PTK2 and PTK2B regulate the NLRP3 inflammasome through different mechanisms

  • PTK2B shows stronger involvement in antiviral pathways

  • Understanding these differences requires careful experimental design with specific antibodies for each protein

What controls and validation strategies are essential when studying PTK2 in different experimental models?

When designing experiments to study PTK2 across different model systems, comprehensive controls and validation strategies are essential:

Validation Matrix for PTK2 Studies:

Researchers should particularly note that validation across species is important, as the search results indicate PTK2 antibodies reactive to human, mouse, and rat . When transitioning between model systems, always verify antibody cross-reactivity and optimize protocols for each specific experimental context.