PXN (Ab-118) Antibody

What is Paxillin and why is the phosphorylated Y118 form important in research?

Paxillin (PXN) is a cytoskeletal protein approximately 65-70 kDa in size that functions critically in actin-membrane attachment at focal adhesions, where cells adhere to the extracellular matrix . The phosphorylation of Paxillin at tyrosine 118 (Y118) represents an important post-translational modification that occurs during cellular signaling events, particularly following growth factor stimulation such as EGF treatment . This specific phosphorylation is a key regulatory event in focal adhesion dynamics, cell migration, and cytoskeletal reorganization. Studying phospho-Paxillin-Y118 enables researchers to investigate dynamic changes in cell adhesion mechanisms and downstream signaling pathways in both normal and pathological conditions.

Which applications can Phospho-Paxillin-Y118 antibodies be reliably used for?

Phospho-Paxillin-Y118 antibodies have been validated for multiple research applications with varying recommended dilutions:

• Western Blotting (WB): Typically used at dilutions of 1:500 to 1:2000

• Enzyme-Linked Immunosorbent Assay (ELISA): Starting concentration of 1 μg/mL, with optimization based on specific assay requirements

• Immunohistochemistry on paraffin-embedded (IHC-P) and frozen sections (IHC-F)

• Immunocytochemistry (ICC): Effective at concentrations around 1-5 μg/mL

• Immunofluorescence (IF): Successfully employed at approximately 5 μg/mL

• Flow Cytometry: Functional at concentrations of about 1 μg per 10^6 cells

The antibody has demonstrated specific binding across these applications, showing distinct bands at approximately 65-70 kDa in Western blot analyses .

What are the recommended sample preparation protocols for detecting phospho-Paxillin?

For optimal detection of phospho-Paxillin-Y118, specific sample preparation protocols are crucial:

For Western Blotting:

• Stimulate cells appropriately (e.g., treat with EGF at 100 ng/mL for 30 minutes following overnight serum starvation)

• Lyse cells using buffers containing phosphatase inhibitors to preserve phosphorylation state

• Load approximately 25-30 μg of protein per lane on 5-20% SDS-PAGE gels

• Use 3-5% BSA rather than milk for blocking to avoid phosphatase activity in milk

• Incubate with the primary antibody overnight at 4°C at dilutions between 1:500-1:2000

For Immunohistochemistry:

• Fix tissues appropriately (preferably with PFA freshly prepared)

• For paraffin sections, perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• Block sections with 10% goat serum

• Incubate with 1-5 μg/mL antibody overnight at 4°C

• Use appropriate detection systems (HRP-conjugated or fluorescent secondary antibodies)

For Immunocytochemistry:

• Fix cells with 4% paraformaldehyde

• Perform membrane permeabilization

• Block with 10% normal goat serum

• Incubate with antibody at 1-5 μg/mL

How should researchers optimize Western blot conditions for phospho-Paxillin-Y118 detection?

To achieve optimal detection of phospho-Paxillin-Y118 in Western blot applications:

• Gel Electrophoresis Parameters:

  • Use 5-20% gradient SDS-PAGE gels

  • Run at 70V through stacking gel and 90V through resolving gel for 2-3 hours

  • Load 25-30 μg protein per lane under reducing conditions

• Transfer Parameters:

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

• Blocking and Antibody Incubation:

  • Block with 3-5% BSA in TBS (not milk) for 1.5 hours at room temperature

  • Incubate with primary antibody (0.5-1 μg/mL) overnight at 4°C

  • Wash with TBS-0.1% Tween (3 times, 5 minutes each)

  • Incubate with HRP-conjugated goat anti-rabbit IgG at 1:5000-1:10000 dilution

• Signal Development:

  • Use enhanced chemiluminescent (ECL) detection systems

  • Optimal exposure time typically ranges from 30 seconds to 2 minutes

• Positive Control:

  • Include lysates from cells treated with EGF (100 ng/mL for 30 minutes after overnight serum starvation)

How can phospho-Paxillin-Y118 antibodies be used to investigate focal adhesion dynamics?

Phospho-Paxillin-Y118 antibodies provide powerful tools for investigating focal adhesion dynamics through several advanced approaches:

• Live Cell Imaging with Immunofluorescence:

  • Combine phospho-Paxillin-Y118 antibody staining with other focal adhesion markers

  • Use time-lapse microscopy following various stimuli to track phosphorylation changes

  • Quantify changes in phospho-Paxillin localization relative to total Paxillin

• Proximity Ligation Assays (PLA):

  • Employ phospho-Paxillin-Y118 antibodies with antibodies against potential binding partners

  • Detect specific protein-protein interactions within focal adhesion complexes

  • Quantify interaction signals under different experimental conditions

• Correlative Analysis with Traction Force Microscopy:

  • Combine phospho-Paxillin-Y118 immunofluorescence with substrate deformation measurements

  • Correlate phosphorylation levels with mechanical force generation at focal adhesions

  • Analyze the relationship between signaling events and mechanical outputs

• Multi-modal Complex Analysis:

  • Investigate the approximately 48-kDa multi-modal complex formed when Paxillin interacts with binding partners like FAT

  • Study how phosphorylation at Y118 affects conformational dynamics in these complexes

  • Combine with structural biology approaches to understand regulatory mechanisms

This integrated approach allows researchers to connect phosphorylation events with functional outcomes in cellular adhesion and migration processes.

What controls should be included when studying phospho-Paxillin-Y118 in experimental designs?

A robust experimental design for phospho-Paxillin-Y118 studies requires several carefully selected controls:

• Phosphorylation State Controls:

  • Positive control: Lysates from cells treated with EGF (100 ng/mL for 30 minutes)

  • Negative control: Lysates from serum-starved cells

  • Phosphatase-treated samples to confirm phospho-specificity

• Antibody Specificity Controls:

  • Peptide competition assays using the immunizing phosphopeptide

  • Parallel blots with antibodies against total Paxillin and phospho-Paxillin

  • Paxillin knockout or knockdown cells (validated with PXN knockout cell lines)

• Loading and Transfer Controls:

  • Housekeeping proteins (β-actin, GAPDH) for equal loading verification

  • Ponceau S staining of membranes to confirm transfer efficiency

  • Stain-free gel technology for total protein normalization

• Cell Type and Species Controls:

  • Include multiple cell lines (HeLa, 293T, HepG2, etc.) to demonstrate consistent detection

  • Test across species (human, mouse, rat) when making cross-species comparisons

• Technical Controls:

  • Secondary antibody-only controls to detect non-specific binding

  • Isotype controls in flow cytometry applications

  • Unstained samples for autofluorescence baseline in fluorescence applications

How do phosphorylation patterns of Paxillin at Y118 differ across cell types and experimental conditions?

Research has revealed distinctive patterns of Paxillin Y118 phosphorylation across different cellular contexts:

• Cell Type-Specific Patterns:

  • Epithelial cells: Show rapid and transient phosphorylation following EGF stimulation

  • Fibroblasts: Display more sustained phosphorylation patterns

  • Cancer cells: Often exhibit elevated basal phosphorylation (particularly in breast and lung cancer cells)

  • Immune cells: Show specialized phosphorylation dynamics during immune synapse formation

• Stimulus-Dependent Responses:

  • Growth factors (EGF, PDGF): Induce rapid phosphorylation within 15-30 minutes

  • ECM engagement: Triggers phosphorylation during initial adhesion formation

  • Mechanical stimuli: Shear stress and substrate stiffness modulate phosphorylation levels

  • Oxidative stress: Alters phosphorylation as part of cellular response mechanisms

• Temporal Dynamics:

  • Initial phosphorylation appears within minutes of stimulation

  • Peak phosphorylation typically occurs at 30-60 minutes post-stimulation

  • Dephosphorylation timing varies significantly by cell type and stimulus

  • Cycling of phosphorylation states correlates with adhesion turnover rates

• Subcellular Localization Differences:

  • Newly formed adhesions: High phospho-Y118 content

  • Mature focal adhesions: Lower phospho-Y118 relative to total Paxillin

  • Leading edge: Enriched phosphorylation during directed migration

  • Perinuclear regions: Occasional phospho-Paxillin accumulation under specific stresses

These diverse patterns highlight the context-dependent regulation of Paxillin phosphorylation and its role in cellular function.

What techniques can be combined with phospho-Paxillin-Y118 antibodies to study conformational dynamics in focal adhesion complexes?

Advanced multi-technique approaches can provide deeper insights into conformational dynamics:

This integrative approach reveals that the Paxillin disordered region undergoes large-scale conformational restriction upon binding to partners like FAT, forming multi-modal complexes with distinct functional properties .

What are common issues when using phospho-Paxillin-Y118 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with phospho-Paxillin-Y118 antibodies:

• Weak or Absent Signal:

  • Problem: Insufficient phosphorylation of target protein

  • Solution: Optimize stimulation conditions (e.g., EGF treatment at 100 ng/mL for 30 minutes after serum starvation)

  • Problem: Phosphatase activity degrading phospho-epitopes

  • Solution: Include phosphatase inhibitors in all buffers and use BSA instead of milk for blocking

• Multiple Bands or High Background:

  • Problem: Non-specific antibody binding

  • Solution: Optimize antibody concentration (typically 0.5-1 μg/mL for WB), increase washing steps

  • Problem: Cross-reactivity with other phospho-proteins

  • Solution: Perform peptide competition assays, increase blocking stringency

• Inconsistent Results Across Experiments:

  • Problem: Variability in phosphorylation states

  • Solution: Standardize cell culture conditions and stimulation protocols

  • Problem: Sample degradation during preparation

  • Solution: Process samples rapidly at cold temperatures with protease/phosphatase inhibitors

• Issues in Immunofluorescence Applications:

  • Problem: Poor signal-to-noise ratio

  • Solution: Optimize fixation method (PFA freshly prepared), increase antibody concentration to 5 μg/mL

  • Problem: Inconsistent staining patterns

  • Solution: Standardize permeabilization conditions, use antigen retrieval for tissue sections

• Variability Between Antibody Lots:

  • Problem: Performance differences between lots

  • Solution: Test and validate each new lot against previous standards, consider recombinant antibodies for consistency

How should researchers validate the specificity of phospho-Paxillin-Y118 antibody signals in their experimental systems?

Thorough validation of phospho-Paxillin-Y118 antibody specificity requires multiple complementary approaches:

• Genetic Validation:

  • Use Paxillin knockout cell lines to confirm signal absence

  • Express wild-type vs. Y118F mutant Paxillin in knockout backgrounds to verify phospho-specificity

  • Employ siRNA knockdown with rescue experiments using phosphorylation site mutants

• Pharmacological Validation:

  • Compare signals before and after treatment with tyrosine phosphatase inhibitors

  • Test signal reduction after treatment with kinase inhibitors that affect pathways upstream of Paxillin

  • Perform lambda phosphatase treatment on control samples

• Peptide Competition:

  • Pre-incubate antibody with increasing concentrations of phospho-Y118 peptide versus non-phosphorylated control peptide

  • Demonstrate dose-dependent signal reduction specifically with phospho-peptide

• Cross-validation with Multiple Methods:

  • Compare results across different techniques (WB, IF, IP-MS)

  • Use alternative phospho-specific antibodies from different vendors/clones

  • Validate with phospho-proteomic mass spectrometry data

• Signal Response Profiling:

  • Demonstrate appropriate signal increases after stimulation with known activators (EGF)

  • Show expected temporal dynamics of phosphorylation and dephosphorylation

  • Verify consistent molecular weight detection (65-70 kDa)

Implementing these validation strategies ensures that experimental observations genuinely reflect Paxillin Y118 phosphorylation rather than artifacts or non-specific signals.

What are the best practices for quantifying phospho-Paxillin-Y118 levels across different experimental conditions?

Accurate quantification of phospho-Paxillin-Y118 requires rigorous methodological approaches:

• Western Blot Quantification:

  • Use total Paxillin normalization rather than housekeeping proteins

  • Calculate phospho-to-total Paxillin ratios for each condition

  • Employ standard curves with known quantities of recombinant phospho-proteins

  • Use digital imaging systems with validated linear detection ranges

  • Perform biological replicates (n≥3) and technical duplicates

• Immunofluorescence Quantification:

  • Capture images using identical acquisition parameters across all samples

  • Measure mean fluorescence intensity within defined focal adhesion regions

  • Normalize phospho-signal to total Paxillin in the same structures

  • Analyze sufficient numbers of cells (typically >30) and adhesions (>100)

  • Use automated, unbiased detection of focal adhesions to prevent selection bias

• Flow Cytometry Analysis:

  • Establish proper gating strategies with isotype controls

  • Quantify median fluorescence intensity rather than percent positive cells

  • Normalize to total Paxillin levels in parallel samples

  • Analyze sufficient events (typically >10,000 cells)

• Statistical Analysis Considerations:

  • Perform appropriate statistical tests based on data distribution

  • Account for multiple comparisons when analyzing multiple experimental conditions

  • Present data showing individual data points alongside means and standard deviations

  • Consider non-parametric tests for small sample sizes

• Experimental Design for Quantification:

  • Include positive controls (EGF-stimulated cells)

  • Add negative controls (unstimulated, phosphatase-treated)

  • Process all samples simultaneously to minimize batch effects

  • Include calibration standards when possible

How can researchers investigate the relationship between Paxillin phosphorylation and conformational dynamics?

Recent advances in structural biology have revealed the complex relationship between Paxillin phosphorylation and its conformational states:

This integrated approach reveals how phosphorylation serves as a molecular switch that modulates Paxillin's conformational landscape and interaction capabilities.

What are the implications of Paxillin Y118 phosphorylation in cancer research and potential therapeutic applications?

Phosphorylation of Paxillin at Y118 has emerged as a significant factor in cancer biology:

• Diagnostic and Prognostic Applications:

  • Elevated phospho-Paxillin-Y118 levels have been detected in various cancer tissues using IHC-P approaches

  • Phosphorylation patterns correlate with tumor invasiveness in certain cancer types

  • Potential biomarker applications for stratifying patient populations

  • Combined analysis with other phospho-proteins may enhance diagnostic accuracy

• Mechanistic Insights in Cancer Progression:

  • Phospho-Paxillin-Y118 regulates focal adhesion dynamics critical for cancer cell invasion

  • Its levels correlate with epithelial-to-mesenchymal transition (EMT) markers

  • Plays roles in invadopodia formation and extracellular matrix degradation

  • Contributes to mechanosensing and adaptation to tumor microenvironment stiffness

• Therapeutic Target Considerations:

  • Direct targeting of phospho-Paxillin-Y118 remains challenging

  • Upstream kinases (FAK, Src) represent more druggable targets

  • Combination approaches targeting multiple nodes in adhesion signaling show promise

  • Biomarker applications for predicting response to kinase inhibitors

• Research Methodology in Cancer Applications:

  • Patient-derived xenograft models for studying phosphorylation in tumor microenvironments

  • Tissue microarray analyses with phospho-Paxillin-Y118 antibodies

  • Correlation of phosphorylation with clinical outcomes and treatment responses

  • Integration with multi-omics data to place Paxillin phosphorylation in broader signaling networks

These applications demonstrate how fundamental research on Paxillin phosphorylation translates into clinically relevant insights and potential therapeutic strategies.

What is the optimal protocol for detecting phospho-Paxillin-Y118 in Western blotting applications?

The following detailed protocol optimizes detection of phospho-Paxillin-Y118 in Western blot applications:

Materials Required:

• Phospho-Paxillin-Y118 primary antibody

• HRP-conjugated anti-rabbit secondary antibody

• Lysis buffer containing phosphatase inhibitors

• SDS-PAGE materials (gels, running buffer)

• Transfer materials (nitrocellulose membrane, transfer buffer)

• BSA for blocking

• Enhanced chemiluminescence (ECL) detection reagents

Protocol:

• Cell Stimulation and Lysis:

  • Serum-starve cells overnight (16-18 hours)

  • Stimulate with EGF (100 ng/mL) for 30 minutes at 37°C

  • Wash cells twice with ice-cold PBS

  • Lyse cells in buffer containing phosphatase inhibitors

  • Sonicate briefly and clarify lysate by centrifugation

• SDS-PAGE Separation:

  • Load 25-30 μg protein per lane on 5-20% gradient gels

  • Run at 70V through stacking gel, then 90V through resolving gel for 2-3 hours

• Protein Transfer:

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

  • Verify transfer efficiency with Ponceau S staining

• Blocking and Antibody Incubation:

  • Block membrane with 3-5% BSA in TBS for 1.5 hours at room temperature

  • Incubate with phospho-Paxillin-Y118 antibody (0.5-1 μg/mL) in 3% BSA/TBS overnight at 4°C

  • Wash 3 times with TBS-0.1% Tween (5 minutes each)

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000-1:10000) for 1.5 hours at room temperature

  • Wash 3 times with TBS-0.1% Tween (5 minutes each)

• Signal Detection:

  • Apply ECL reagents according to manufacturer's instructions

  • Expose to X-ray film or use digital imaging system

  • Optimal exposure time typically ranges from 30 seconds to 2 minutes

• Stripping and Reprobing (for total Paxillin):

  • Strip membrane using commercial stripping buffer

  • Re-block with 3-5% BSA/TBS

  • Reprobe with total Paxillin antibody to calculate phospho/total ratios

Expected Results:

• Phospho-Paxillin-Y118 should appear as a distinct band at approximately 65-70 kDa

• EGF-stimulated samples should show increased signal intensity compared to unstimulated controls

• Multiple cell types can be analyzed including HeLa, 293T, HepG2, and others

What are the recommended protocols for immunofluorescence detection of phospho-Paxillin-Y118 in adherent cells?

This detailed protocol maximizes immunofluorescence detection of phospho-Paxillin-Y118:

Materials Required:

• Phospho-Paxillin-Y118 primary antibody

• Fluorophore-conjugated secondary antibody

• Glass coverslips or chamber slides

• Paraformaldehyde (PFA) fixative (freshly prepared)

• Permeabilization buffer

• Blocking solution (10% normal goat serum)

• Mounting medium with DAPI

• Phosphate-buffered saline (PBS)

Protocol:

• Cell Preparation:

  • Culture cells on glass coverslips or chamber slides

  • For optimal focal adhesion visualization, plate cells on fibronectin-coated (10 μg/mL) surfaces

  • Allow cells to adhere for 24-48 hours to form mature focal adhesions

• Stimulation (Optional):

  • Serum-starve cells overnight (16-18 hours)

  • Stimulate with EGF (100 ng/mL) for 15-30 minutes

• Fixation and Permeabilization:

  • Fix cells with freshly prepared 4% PFA for 15 minutes at room temperature

  • Wash 3 times with PBS (5 minutes each)

  • Permeabilize with 0.1% Triton X-100 in PBS for 5 minutes

  • Wash 3 times with PBS (5 minutes each)

• Blocking and Antibody Incubation:

  • Block with 10% normal goat serum in PBS for 1 hour at room temperature

  • Incubate with phospho-Paxillin-Y118 antibody (5 μg/mL) in blocking buffer overnight at 4°C

  • Wash 3 times with PBS (5 minutes each)

  • Incubate with fluorophore-conjugated anti-rabbit secondary antibody (e.g., DyLight 488-conjugated goat anti-rabbit IgG, 1:100-1:200) for 1 hour at room temperature

  • Wash 3 times with PBS (5 minutes each)

• Counterstaining and Mounting:

  • Counterstain with DAPI to visualize nuclei

  • Mount coverslips on slides using anti-fade mounting medium

  • Seal edges with nail polish and store at 4°C protected from light

• Imaging:

  • Visualize using a fluorescence microscope with appropriate filter sets

  • For detailed focal adhesion analysis, use confocal or super-resolution microscopy

  • Capture multiple fields (>10) for quantitative analysis

  • Use consistent exposure settings across all experimental conditions

Expected Results:

• Phospho-Paxillin-Y118 typically localizes as distinct punctate structures at the cell periphery, corresponding to focal adhesions

• EGF-stimulated cells should show increased phospho-Paxillin-Y118 signal intensity

• Colocalization with other focal adhesion markers can be assessed in multi-channel imaging

How can phospho-Paxillin-Y118 antibodies be utilized in high-content screening applications?

High-content screening with phospho-Paxillin-Y118 antibodies enables large-scale analyses of adhesion dynamics:

Materials and Equipment Required:

• Phospho-Paxillin-Y118 antibody

• Fluorophore-conjugated secondary antibodies

• Multi-well plates (96- or 384-well format)

• Automated liquid handling system

• High-content imaging platform

• Image analysis software

• Compound libraries or siRNA/CRISPR libraries

Protocol:

• Assay Setup:

  • Seed cells in multi-well plates at optimized density (typically 5,000-10,000 cells/well for 96-well format)

  • For matrix studies, coat wells with different ECM proteins (fibronectin, collagen, laminin)

  • Allow cells to adhere for 24-48 hours

• Treatment:

  • Apply compounds, siRNAs, or CRISPR libraries using automated liquid handling

  • Include positive controls (EGF treatment) and negative controls (vehicle, non-targeting siRNA)

  • Incubate for predetermined time periods (acute: 15-60 minutes; chronic: 24-72 hours)

• Immunofluorescence Staining:

  • Fix cells with 4% PFA for 15 minutes

  • Permeabilize with 0.1% Triton X-100 for 5 minutes

  • Block with 10% normal goat serum

  • Incubate with phospho-Paxillin-Y118 antibody (5 μg/mL) overnight at 4°C

  • Apply fluorescent secondary antibody

  • Counterstain nuclei with DAPI

  • For multiplexed analysis, include additional markers (F-actin, total Paxillin, other focal adhesion proteins)

• Automated Imaging:

  • Acquire images using high-content imaging system

  • Capture multiple fields per well (typically 9-16)

  • Use appropriate magnification (20-40×) for focal adhesion resolution

  • Implement autofocus for consistent image quality

• Image Analysis:

  • Develop analysis pipeline including:

    • Cell segmentation based on nuclear and cytoplasmic markers

    • Focal adhesion identification using phospho-Paxillin-Y118 signal

    • Quantification of parameters: number, size, intensity, and distribution of phospho-Paxillin-positive structures

    • Cell morphology metrics (area, perimeter, shape factor)

    • Integrated intensity of phospho-Paxillin-Y118 normalized to total Paxillin

• Data Analysis:

  • Apply quality control metrics to remove outliers

  • Normalize data to internal controls

  • Perform statistical analysis across treatment conditions

  • Generate dose-response curves for compound screens

  • Identify hits based on predetermined thresholds

This high-throughput approach enables screening of thousands of conditions to identify modulators of Paxillin phosphorylation and focal adhesion dynamics, with applications in drug discovery and mechanistic studies of cell adhesion regulation.