Phospho-PXN (Tyr31) Antibody

What is paxillin and why is its Tyr31 phosphorylation significant in cellular research?

Paxillin (PXN) is a 76-kDa focal adhesion protein first identified in Rous sarcoma virus-transformed chick embryo fibroblasts. The name derives from the Latin 'paxillus' meaning 'small stake' or 'peg,' reflecting its role in tethering membrane structures at focal adhesions . Paxillin serves as a critical scaffolding protein that localizes to focal adhesions at the ends of actin-containing stress fibers in non-transformed cells and coordinates various signaling networks .

Tyrosine 31 phosphorylation represents one of the key regulatory modifications of paxillin, particularly in the context of:

• Cell migration and invasion processes

• Integrin-mediated signaling pathways

• Cytoskeletal reorganization during cell motility

• Cancer cell metastasis and proliferation

Phosphorylation at Tyr31 is typically mediated by Src family kinases and FAK (Focal Adhesion Kinase), creating binding sites for SH2 domain-containing proteins that further propagate downstream signaling cascades.

What are the fundamental differences between polyclonal and monoclonal Phospho-PXN (Tyr31) antibodies?

These two antibody types offer distinct advantages and limitations for phospho-paxillin research:

For experiments requiring absolute specificity and reproducibility, monoclonal antibodies offer advantages, while polyclonals may provide better sensitivity for detecting low-abundance phosphorylated paxillin in complex samples.

How can researchers validate the specificity of Phospho-PXN (Tyr31) antibodies?

Rigorous validation is essential when working with phospho-specific antibodies. Recommended validation procedures include:

• Phosphatase treatment control: Split your sample and treat half with lambda phosphatase to remove phosphorylation. A specific phospho-antibody will show signal only in the untreated sample.

• Mutation analysis: Express wild-type paxillin alongside a Y31F mutant (tyrosine replaced with non-phosphorylatable phenylalanine). The phospho-specific antibody should only recognize the wild-type protein when phosphorylated.

• Peptide competition assay: Pre-incubate antibody with excess phosphorylated peptide (T-P-Y(p)-S-Y) and separately with non-phosphorylated peptide. Signal should be blocked only by the phosphopeptide.

• Kinase activation/inhibition: Treat cells with kinase activators (e.g., EGF, PDGF for tyrosine kinases) or specific inhibitors (e.g., Src family inhibitors). Phospho-signal should increase with activation and decrease with inhibition.

• Multiple antibody comparison: Compare results from different antibodies targeting the same phospho-site (e.g., both polyclonal and monoclonal anti-Phospho-PXN Tyr31).

This multi-pronged approach ensures that observed signals genuinely represent Tyr31 phosphorylation rather than non-specific binding or artifacts.

What are the optimal storage conditions for maintaining Phospho-PXN (Tyr31) antibody activity?

Proper storage is critical for preserving antibody functionality:

• Store antibodies at -20°C in small working aliquots to minimize freeze-thaw cycles .

• For longer-term storage, -80°C provides enhanced stability .

• Avoid repeated freeze-thaw cycles, which can lead to protein denaturation and reduced activity .

• Most commercial preparations are supplied in stabilizing buffer containing glycerol (typically 50%), which prevents freezing solid and reduces damage from ice crystal formation .

• Standard buffer composition includes PBS (without Mg²⁺ and Ca²⁺), pH 7.4, containing 150mM NaCl, 0.02% sodium azide, and 50% glycerol .

• When in use, keep antibodies on ice to minimize degradation.

• For reconstituted lyophilized antibodies, follow manufacturer's specific instructions for reconstitution volume and storage.

Following these practices can significantly extend the functional lifetime of these valuable research reagents.

What sample preparation protocols optimize detection of phosphorylated paxillin in various experimental conditions?

Effective sample preparation is crucial for phosphoprotein detection:

Cell Lysis Buffer Composition:

• Base buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40/Triton X-100

• Protease inhibitors: PMSF (1 mM), aprotinin (10 μg/ml), leupeptin (10 μg/ml)

• Critical phosphatase inhibitors: Sodium orthovanadate (1 mM), sodium fluoride (10 mM), β-glycerophosphate (10 mM), and EDTA (5 mM)

Recommended Protocol:

• When harvesting adherent cells, avoid prolonged trypsinization which can alter phosphorylation status.

• Rapidly lyse cells directly in dish by adding ice-cold lysis buffer after washing with cold PBS.

• Maintain constant low temperature during all processing steps to inhibit phosphatase activity.

• Clarify lysates by centrifugation (14,000 × g, 15 minutes, 4°C).

• Determine protein concentration using a detergent-compatible assay.

• Add 5× Laemmli buffer and heat at 95°C for 5 minutes.

• Load 20-50 μg total protein per lane for western blotting.

Tissue Sample Considerations:

• Flash-freeze tissues immediately in liquid nitrogen and store at -80°C until processing.

• Homogenize tissue in lysis buffer containing phosphatase inhibitors using a mechanical homogenizer.

• Process tissue samples rapidly to minimize dephosphorylation.

These protocols ensure maximal preservation of phosphorylation status for accurate assessment of paxillin Tyr31 phosphorylation levels.

What are the recommended working dilutions and conditions for Phospho-PXN (Tyr31) antibodies in Western blotting?

Optimal working conditions vary by antibody source, but general guidelines include:

Recommended Dilutions:

• For Western blotting: 1:500 to 1:1000 dilution range is typically optimal

• For ELISA: Similar dilution range (1:500 to 1:1000) is effective

Western Blotting Protocol Optimization:

• Transfer conditions: Use PVDF membrane (0.45 μm) for better protein retention.

• Blocking: 5% BSA in TBST (not milk, which contains phosphatases) for 1 hour at room temperature.

• Primary antibody incubation: Dilute antibody in 5% BSA-TBST and incubate overnight at 4°C.

• Washing: 4 × 5-minute washes with TBST.

• Secondary antibody: Anti-rabbit HRP (for polyclonal) or anti-mouse HRP (for monoclonal) at 1:5000 in 5% BSA-TBST for 1 hour at room temperature .

• Detection: Enhanced chemiluminescence systems provide optimal signal-to-noise ratio.

Important Considerations:

• Always perform titration experiments to determine optimal concentration for your specific experimental system.

• Include positive controls (EGF-stimulated cells) and negative controls (phosphatase-treated lysates).

• For quantitative analysis, ensure detection falls within linear range of assay.

These conditions maximize sensitivity while minimizing background for phospho-paxillin detection.

How can Phospho-PXN (Tyr31) antibodies be used to monitor cell migration and focal adhesion dynamics?

Phospho-PXN (Tyr31) antibodies serve as powerful tools for investigating cellular migration mechanisms:

Immunofluorescence Applications:

• Focal adhesion turnover analysis: Track phospho-paxillin as a marker of adhesion formation and disassembly.

• Co-localization studies: Combine with other focal adhesion markers (vinculin, FAK) to map spatial relationships during migration.

• Live-cell imaging: Use in conjunction with GFP-paxillin to correlate total vs. phosphorylated protein dynamics.

Migration Assay Integration:

• Wound healing assays: Fix cells at various timepoints post-scratch and stain for phospho-paxillin to visualize directional activation.

• Transwell migration: Quantify phospho-paxillin levels in migrating vs. non-migrating cell populations.

• 3D matrix invasion: Compare phospho-paxillin distribution in 2D vs. 3D environments to understand dimensional effects on signaling.

Advanced Analytical Approaches:

• FRET-based sensors: Combine with engineered paxillin FRET sensors to measure phosphorylation in real-time.

• Ratiometric imaging: Calculate phospho-paxillin:total paxillin ratios to normalize for expression level differences.

• High-content screening: Develop phospho-paxillin-based phenotypic screens for compounds affecting cell migration.

When properly implemented, these approaches yield valuable insights into the spatiotemporal regulation of focal adhesion dynamics during cell migration.

How does phosphorylation at Tyr31 interact with other post-translational modifications on paxillin?

Paxillin undergoes complex, multi-site post-translational modifications that create a sophisticated regulatory network:

Interplay Between Phosphorylation Sites:

• Tyr31/Tyr118 co-regulation: These sites often show coordinated phosphorylation, both being Src/FAK targets. Detection with dual phospho-antibodies can reveal hierarchical phosphorylation patterns.

• Serine phosphorylation crosstalk: Phosphorylation at Ser273 by PAK1 can influence Tyr31 accessibility. Pre-treatment with PKC activators (PMA) before probing for Tyr31 phosphorylation can reveal sequential dependencies.

• Hierarchical modification: In some contexts, one modification creates recognition sites for subsequent enzymes (priming).

Multi-PTM Detection Strategies:

• Sequential immunoprecipitation: First IP with anti-phospho-Tyr31, then probe with antibodies against other modifications.

• Mass spectrometry: Identify comprehensive modification patterns using phosphopeptide enrichment combined with LC-MS/MS.

• Phos-tag™ gels: Resolve multiply-phosphorylated forms of paxillin based on phosphate number/position before western blotting.

Functional Consequences of Combined Modifications:

• Different phosphorylation combinations create distinct protein-interaction surfaces

• Modification patterns dictate subcellular localization and turnover rates

• Temporal sequences of modifications regulate adhesion assembly/disassembly cycles

Understanding these complex interactions requires simultaneous monitoring of multiple phosphorylation events, often combining phospho-specific antibodies with other analytical techniques.

What are the latest approaches for generating highly specific monoclonal phospho-specific antibodies against paxillin?

Recent developments have transformed phospho-specific antibody generation:

In Vitro Selection Methods:

A particularly innovative approach involves using full-length phosphorylated proteins as antigens :

• Express full-length paxillin in bacteria with a polyhistidine tag

• Purify using Ni-NTA resin

• Enzymatically phosphorylate by exposure to kinase-containing mitotic cell extract

• Confirm modification via mobility shift assay

• Use the phosphorylated full-length protein for direct antibody selection using phage display libraries

This approach offers several advantages over traditional peptide immunization methods:

• Allows selection of antibodies against the naturally folded protein

• Enables discovery of antibodies against multiple phospho-epitopes simultaneously

• Favors selection of antibodies against major physiological phosphorylation sites

• Circumvents protein degradation, dephosphorylation, and antigen processing issues that occur in animal immunization

Recombinant Antibody Engineering:

• Phage display technology: Enables selection of highly specific single-chain variable fragments (scFvs)

• Affinity maturation: Directed evolution approaches to enhance specificity and affinity

• Epitope-focused libraries: Creation of antibody libraries specifically designed to recognize phosphotyrosine in different sequence contexts

These advanced methods can yield phospho-specific antibodies with superior performance characteristics in shorter timeframes than traditional approaches.

What methodological considerations are important when using Phospho-PXN (Tyr31) antibodies in multiplex immunostaining?

Multiplexed detection systems require careful optimization:

Technical Considerations:

• Antibody compatibility: Ensure primary antibodies are from different host species or use directly conjugated antibodies to avoid cross-reactivity.

• Sequential staining protocol: For same-species antibodies, use tyramide signal amplification with sequential microwave treatment to allow multiple rounds of staining.

• Signal separation: Carefully select fluorophores with minimal spectral overlap.

• Antigen retrieval optimization: Different phospho-epitopes may require distinct retrieval conditions, requiring compromise for multiplex detection.

Recommended Multiplexing Approaches:

• Phospho-Tyr31 + total paxillin: Combine rabbit anti-phospho-Tyr31 with mouse anti-total paxillin to calculate phosphorylation ratios within individual cells.

• Multi-phosphosite analysis: Pair phospho-Tyr31 (rabbit) with phospho-Tyr118 (mouse) and phospho-Ser273 (goat) to visualize phosphorylation patterns.

• Pathway integration: Combine phospho-Tyr31 with phospho-FAK and phospho-Src antibodies to map kinase-substrate relationships.

Controls for Multiplex Validation:

• Single antibody staining controls to confirm specificity

• Blocking peptide controls to verify epitope specificity

• Signal bleed-through controls to ensure proper spectral separation

• Phosphatase-treated samples as negative controls

These approaches allow researchers to extract maximum information from limited samples while maintaining quantitative accuracy.

How can researchers address non-specific binding when using Phospho-PXN (Tyr31) antibodies?

Non-specific binding represents a common challenge with phospho-specific antibodies:

Common Sources of Non-Specificity:

• Cross-reactivity with related phospho-epitopes: Similar phosphotyrosine-containing sequences in other proteins

• Binding to non-phosphorylated epitopes: Incomplete removal of antibodies recognizing the non-phosphorylated sequence

• Secondary antibody cross-reactivity: Especially problematic in multiplexed applications

Optimization Strategies:

• Antibody purification: Using affinity chromatography with non-phosphopeptide to remove antibodies recognizing the non-phosphorylated backbone

• Blocking optimization: Using 5% BSA rather than milk (which contains phosphatases)

• Peptide competition: Pre-incubate antibody with excess phosphopeptide to block specific binding sites

• Titration experiments: Systematically test dilution series to find optimal signal-to-noise ratio

• Alternative antibody formats: Consider monoclonal antibodies if polyclonals show high background

Validation Controls:

• Y31F mutant expression: A non-phosphorylatable mutant provides the ideal negative control

• siRNA knockdown: Reduced signal with paxillin knockdown confirms specificity

• Knockout cells/tissues: Samples lacking paxillin should show no signal

Implementing these approaches systematically can significantly improve signal specificity and experimental reliability.

What approaches can resolve contradictory results between Phospho-PXN (Tyr31) antibody detection and functional assays?

Discrepancies between phospho-antibody detection and functional outcomes require systematic troubleshooting:

Common Causes of Discrepancies:

• Temporal dynamics: Phosphorylation may be transient while functional effects persist

• Threshold effects: Functional changes may require specific phosphorylation levels

• Compartmentalization: Global phosphorylation measurements may miss localized changes

• Combinatorial modifications: Functional outcomes often depend on multiple modifications

• Technical artifacts: Sample preparation may alter phosphorylation status

Resolution Strategies:

• Time-course experiments: Detailed temporal mapping of both phosphorylation and functional outcomes

• Dose-response relationships: Correlate phosphorylation levels with functional readouts across stimulation ranges

• Cellular fractionation: Separate cytosolic, membrane, nuclear, and cytoskeletal fractions before analysis

• Kinase/phosphatase manipulation: Use specific inhibitors/activators to establish causality

• Phosphomimetic/phosphodeficient mutations: Y31E (phosphomimetic) and Y31F (phosphodeficient) mutants can help establish causal relationships

Advanced Analytical Approaches:

• Proximity ligation assays: Detect specific protein-protein interactions dependent on phosphorylation

• Single-cell analysis: Correlate phosphorylation with function at individual cell level

• Quantitative phosphoproteomics: Obtain comprehensive view of phosphorylation networks

These approaches help bridge the gap between biochemical observations and functional outcomes.

How can quantitative analysis of Phospho-PXN (Tyr31) levels be standardized across experiments?

Standardization is essential for reliable quantitative comparisons:

Internal Controls and Normalization:

• Total protein normalization: Measure phospho-Tyr31 signal relative to total paxillin levels

• Housekeeping proteins: Use stable references like GAPDH or β-actin as loading controls

• Phosphorylation standards: Include lysates from cells treated with known stimulators (EGF, pervanadate) as positive controls

• Standardized positive control: Create a large batch of phosphorylated cell lysate, aliquot and freeze as reference standard

Quantification Methodologies:

• Densitometry guidelines: Ensure detection within linear range of assay

• Fluorescent western blotting: Provides wider linear range than chemiluminescence

• ELISA-based quantification: Sandwich ELISA with capture antibody against total paxillin and detection with phospho-Tyr31

• Flow cytometry: For single-cell quantification in heterogeneous populations

Technical Validation Parameters:

• Intra-assay coefficient of variation (<10%)

• Inter-assay coefficient of variation (<15%)

• Lower limit of detection and quantification

• Dose-response linearity

Reporting Standards:

• Always report both raw and normalized values

• Include detailed methodology for phospho-signal quantification

• Provide sample size and statistical analysis methods

• Indicate antibody source, catalog number, and lot number

Following these standardization practices enables meaningful cross-experimental comparisons and improves research reproducibility.

What are the advantages and limitations of using bacterially expressed phosphorylated proteins for antibody selection?

This innovative approach offers distinct advantages and challenges:

Advantages:

• Full-length protein context: Allows selection of antibodies against the complete, properly folded protein rather than short peptides

• Multiple epitope targeting: Enables simultaneous selection of antibodies against different phosphorylation sites

• Natural phosphorylation patterns: Using kinase-containing cell extracts creates physiologically relevant modifications

• Rapid timeline: The entire process can be completed in less than one week

• Resource efficiency: Avoids costly and time-consuming animal immunization protocols

Limitations:

• Bacterial expression challenges: Some eukaryotic proteins express poorly or form inclusion bodies in bacteria

• Post-translational modification differences: Bacteria lack many eukaryotic PTMs that might influence epitope structure

• In vitro phosphorylation efficiency: May not achieve complete phosphorylation at all relevant sites

• Potential for non-physiological modifications: Cell extracts contain multiple kinases that could create non-native phosphorylation patterns

• Protein stability concerns: Some phosphorylated proteins may be unstable during the selection process

Recommended Applications:

This approach is particularly valuable for:

• Rapidly generating multiple phospho-specific antibodies against a single protein

• Targeting physiologically significant phosphorylation sites without prior site mapping

• Developing antibodies against difficult-to-synthesize phosphopeptides

• Situations requiring multiple phospho-specific antibodies for comprehensive pathway analysis

When properly implemented, this method can significantly accelerate phosphoproteomics research by providing high-quality, site-specific phospho-antibodies.

How can researchers differentiate between true signal and artifacts when analyzing Phospho-PXN (Tyr31) localization in microscopy?

Accurate interpretation of immunofluorescence data requires rigorous controls and optimization:

Common Artifacts in Phospho-Immunostaining:

• Fixation artifacts: Different fixation methods can alter phosphoepitope preservation

• Non-specific binding: Secondary antibody cross-reactivity or primary antibody off-target binding

• Autofluorescence: Cellular components or fixatives causing background signal

• Bleed-through: Spectral overlap between fluorophores in multi-channel imaging

• Phosphatase activity: Loss of phosphorylation during sample processing

Validation Controls:

• Y31F mutant-expressing cells: Should show minimal or no signal

• Phosphatase treatment: Pre-treatment of fixed cells with lambda phosphatase

• Competition with phospho-peptide: Pre-incubation of antibody with excess specific phosphopeptide

• Kinase activation/inhibition: Treatment with activators (e.g., EGF) or inhibitors of upstream kinases

• Multiple antibody comparison: Use different antibodies targeting the same phospho-site

Optimized Protocol Elements:

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature preserves most phosphoepitopes

• Permeabilization: Gentle detergent treatment (0.1% Triton X-100, 5 minutes)

• Blocking: 1-3% BSA with 10% normal serum from secondary antibody host species

• Antibody incubation: Overnight at 4°C for primary antibody at validated dilution

• Mounting: Anti-fade mountant without DAPI to prevent photobleaching

Advanced Imaging Considerations:

• Use confocal microscopy to improve signal localization

• Implement deconvolution algorithms to enhance signal-to-noise ratio

• Employ quantitative co-localization analysis with other focal adhesion markers

• Consider super-resolution techniques for precise localization within focal adhesion structures

These approaches enable confident interpretation of phospho-paxillin localization patterns in diverse experimental contexts.

How can phosphatase inhibitors be effectively used when working with Phospho-PXN (Tyr31) antibodies?

Phosphatase inhibition is critical for preserving phosphorylation status:

Optimal Phosphatase Inhibitor Cocktail:

Implementation Guidelines:

• Fresh preparation: Prepare sodium orthovanadate solution fresh and properly activate by boiling/pH adjustment

• Temperature control: Keep samples cold (on ice) throughout processing

• Rapid processing: Minimize time between cell lysis and protein denaturation

• Pre-treatment considerations: For adherent cells, add phosphatase inhibitors directly to culture media before lysis

• Storage precautions: Include phosphatase inhibitors in storage buffers for long-term sample preservation

Application-Specific Recommendations:

• Cell lysis: Include complete phosphatase inhibitor cocktail in lysis buffer

• Immunoprecipitation: Add inhibitors to all buffers throughout procedure

• Tissue processing: Pre-treat tissues with inhibitors before homogenization

• In vitro assays: Include inhibitors when studying kinase activities to prevent substrate dephosphorylation

Proper phosphatase inhibition is essential for accurate assessment of the phosphorylation status of transient modifications like Tyr31 phosphorylation.

What are the technical considerations when designing cellular experiments to study phospho-paxillin dynamics?

Experimental design significantly impacts the quality of phospho-paxillin data:

Cell Culture Considerations:

• Cell density effects: Confluency alters cell-cell contacts and focal adhesion formation

• Substrate composition: Different ECM proteins (fibronectin, collagen, laminin) induce distinct phosphorylation patterns

• Serum starvation: Required to establish baseline phosphorylation before stimulation

• Cell passage number: Higher passages may show altered signaling responses

• Cell detachment methods: Trypsinization can alter phosphorylation; mechanical scraping or enzyme-free solutions preferred

Stimulation Protocols:

• Growth factor treatments:

  • EGF (50-100 ng/ml): Rapid induction within 5-15 minutes

  • PDGF (25-50 ng/ml): Sustained response over 30-60 minutes

  • Serum (10%): Complex response involving multiple pathways

• Adhesion dynamics:

  • Suspension-reattachment assays: Trypsinize cells, hold in suspension, then plate on ECM

  • Micropatterned substrates: Control focal adhesion size and distribution

  • Mechanical stimulation: Substrate stretching induces rapid phosphorylation changes

• Inhibitor studies:

  • Src inhibitors (PP2, dasatinib): Block primary kinase for Tyr31

  • FAK inhibitors (PF-573228): Interrupt FAK-Src signaling complex

  • Actin cytoskeleton disruptors (cytochalasin D): Reveal cytoskeleton-dependence

Time-Course Considerations:

• Rapid events: Sample at 0, 2, 5, 15, 30 minutes

• Extended dynamics: Include 1, 2, 4, 8, 24 hour timepoints

• Consider both phosphorylation and dephosphorylation kinetics

Data Collection Framework:

• Always include both phospho-specific and total protein measurements

• Quantify multiple parameters (phosphorylation intensity, focal adhesion size, number)

• Consider single-cell analysis to account for heterogeneity

• Implement appropriate statistical methods for time-course data