Phospho-PXN (Tyr118) Antibody

What is Phospho-PXN (Tyr118) Antibody and what does it detect?

Phospho-PXN (Tyr118) antibody specifically recognizes paxillin protein only when phosphorylated at tyrosine residue 118. Paxillin is a 68 kDa focal adhesion protein involved in actin-membrane attachment at sites of cell adhesion to the extracellular matrix (focal adhesions) . The antibody is typically generated by immunizing rabbits with synthetic phosphopeptides corresponding to amino acid sequences surrounding the Tyr118 residue of human paxillin .

These antibodies are crucial for studying phosphorylation-dependent signaling events in focal adhesions because tyrosine 118 represents one of the major phosphorylation sites in paxillin. The phosphorylation at this position occurs in response to growth factors, neuropeptides, and integrin engagement, making it a key regulatory event in multiple cellular processes including cell migration and adhesion dynamics .

What species reactivity can I expect with Phospho-PXN (Tyr118) Antibody?

Most commercially available Phospho-PXN (Tyr118) antibodies demonstrate confirmed reactivity with human, mouse, and rat samples as indicated in multiple product specifications . Some antibodies also show cross-reactivity with additional species:

The high degree of conservation in the tyrosine 118 region of paxillin across various species enables this broad reactivity profile. When planning experiments with less common model organisms, verifying reactivity through preliminary testing is recommended .

What are the common applications for Phospho-PXN (Tyr118) Antibody?

The primary applications for Phospho-PXN (Tyr118) Antibody include:

• Western Blotting (WB): This is the most commonly validated application across all manufacturers, typically using a dilution range of 1:500-1:2000 . The antibody detects a band at approximately 68 kDa corresponding to phosphorylated paxillin in stimulated samples.

• Enzyme-Linked Immunosorbent Assay (ELISA): Several antibodies are validated for ELISA applications with recommended dilutions around 1:10000 .

• Immunohistochemistry (IHC): Some formats are validated for visualization of phosphorylated paxillin in tissue sections .

For optimal results, each application requires specific optimization of antibody dilution and sample preparation protocols to ensure specific detection of the phosphorylated form. Most manufacturers recommend verification of specificity using appropriate controls, such as treating duplicate samples with phosphatase or using competing phosphopeptides .

What are the optimal methods for inducing paxillin Tyr118 phosphorylation in cell culture?

To effectively induce paxillin Tyr118 phosphorylation for antibody validation or experimental studies, several approaches have been demonstrated effective:

• Growth factor stimulation: Treatment with Epidermal Growth Factor (EGF) has been widely used to induce paxillin phosphorylation in multiple cell types, particularly in A431 cells (human epidermoid carcinoma cells) . Western blot analysis from multiple antibody suppliers confirms robust phosphorylation at Tyr118 following EGF treatment.

• Tyrosine phosphatase inhibition: Treatment with pervanadate effectively preserves phosphorylation by inhibiting dephosphorylation, as demonstrated in antibody validation studies . This approach is particularly useful when studying basal phosphorylation levels that might otherwise be rapidly dephosphorylated.

• TGF-β treatment: Particularly effective in epithelial cells, as demonstrated in studies using normal mouse mammary gland (NMuMG) cells with EGFP-tagged paxillin .

• Integrin activation: Plating cells on extracellular matrix components such as fibronectin can induce paxillin phosphorylation through integrin-mediated signaling pathways .

The optimal induction method depends on cell type and the specific signaling pathway under investigation. Including appropriate positive controls such as A431 cells treated with EGF is recommended for validation of antibody specificity and experimental setup .

What is the best sample preparation protocol for detecting phospho-paxillin (Tyr118) in Western blotting?

For optimal detection of phospho-paxillin (Tyr118) in Western blotting, the following comprehensive protocol incorporates best practices from multiple sources:

• Cell lysis buffer composition:

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

  • Phosphatase inhibitors: Sodium orthovanadate (Na₃VO₄), sodium fluoride (NaF), β-glycerophosphate

  • Protease inhibitors: Complete protease inhibitor cocktail

  • Additional components: 0.5% BSA and glycerol (as included in some commercial preparations)

• Sample handling:

  • Maintain samples at 4°C throughout processing to preserve phosphorylation status

  • Process samples quickly after stimulation (e.g., EGF treatment or cell adhesion)

  • Avoid repeated freeze-thaw cycles of lysates which can degrade phospho-epitopes

• Gel electrophoresis and transfer:

  • Use fresh polyacrylamide gels (8-10%) for optimal separation

  • Load 20-50 μg of total protein per lane

  • Transfer to PVDF membrane (preferred over nitrocellulose for phospho-proteins)

  • Block with 5% BSA in TBST rather than milk (which contains phosphatases)

• Antibody incubation:

  • Dilute primary antibody 1:500-1:2000 in 5% BSA/TBST as recommended by multiple manufacturers

  • Incubate overnight at 4°C with gentle agitation for optimal binding

  • Use appropriate HRP-conjugated secondary antibodies with careful optimization of dilution

• Controls and validation:

  • Include positive control (e.g., A431 cells treated with EGF)

  • Consider including a phosphatase-treated sample as negative control

  • Use peptide competition with phosphorylated and non-phosphorylated peptides to confirm specificity

This protocol maximizes preservation of phosphorylation status while minimizing background signal, allowing for reliable detection of phospho-paxillin (Tyr118).

How can I distinguish between specific and non-specific signals when using Phospho-PXN (Tyr118) Antibody?

Ensuring specificity of phospho-paxillin (Tyr118) detection requires implementing several validation approaches:

• Essential controls:

  • Dephosphorylation control: Treat duplicate samples with alkaline phosphatase before electrophoresis to confirm signal loss

  • Peptide competition: Pre-incubate antibody with phosphorylated peptide immunogen to block specific binding. Western blot analysis showing successful blocking of signal with phosphopeptide has been demonstrated by multiple antibody suppliers

  • Negative control: Use unstimulated cells where paxillin phosphorylation is minimal, or cells treated with kinase inhibitors (FAK or Src inhibitors)

• Validation approaches:

  • Molecular weight verification: Confirm signal at 68-70 kDa (paxillin's expected molecular weight)

  • Stimulation-dependent signal: Compare samples with and without stimulation (e.g., EGF, pervanadate)

  • Signal intensity correlation: Verify increased signal intensity corresponds with increased stimulation duration or concentration

• Western blot specificity checks:

  • The phospho-signal should disappear with phosphatase treatment but remain present in untreated samples

  • Signal should be blocked by competition with the phosphorylated peptide but not with a non-phosphorylated version of the same peptide

  • The antibody should recognize paxillin only when phosphorylated at tyrosine 118, not under basal conditions

• Quantification methods:

  • Always normalize phospho-signal to total paxillin levels using a non-phospho-specific paxillin antibody

  • Compare the ratio of phospho-paxillin to total paxillin across experimental conditions for accurate relative quantification

These validation steps, as demonstrated in multiple antibody characterization studies, ensure that observed signals represent genuine phospho-paxillin (Tyr118) rather than cross-reactivity with other phospho-proteins .

Which kinases are responsible for paxillin Tyr118 phosphorylation in different cellular contexts?

The phosphorylation of paxillin at Tyr118 involves multiple kinases depending on cell type and stimulus. Based on detailed protein database information and experimental evidence:

The UniProt database entry for paxillin (P49023) confirms these kinase relationships for the Tyr118 site . The phosphorylation pattern depends on the cellular context:

• Adhesion-dependent phosphorylation: Primarily mediated by FAK and Src family kinases during integrin engagement and focal adhesion formation .

• Growth factor-dependent phosphorylation: Often involves receptor tyrosine kinase signaling cascades that activate Src, as seen in EGF stimulation experiments used to validate multiple Phospho-PXN (Tyr118) antibodies .

• Mechanical stimulation: FAK activation following mechanical force application can lead to paxillin phosphorylation at Tyr118, important in mechanotransduction studies.

Understanding the kinase responsible in a specific model system is crucial for appropriate experimental design and interpretation of phospho-paxillin (Tyr118) data .

How does phosphorylation at Tyr118 interact with other post-translational modifications of paxillin?

Paxillin undergoes multiple post-translational modifications that interact with Tyr118 phosphorylation in complex ways. The comprehensive post-translational modification database from Affinity Biosciences reveals numerous modification sites on paxillin :

• Additional phosphorylation sites that interact with Tyr118:

  • Tyr31 phosphorylation: Often co-occurs with Tyr118 phosphorylation, also mediated by FAK, ABL1, and PTK6

  • Tyr88 phosphorylation: Mediated by Src, potentially affecting Tyr118 phosphorylation

  • Ser126 phosphorylation: Mediated by NLK (Nemo-like kinase), occurs near Tyr118

  • Ser130 phosphorylation: Mediated by MAPK3, in proximity to Tyr118

• Ubiquitination sites near Tyr118:

  • K123 and K125 ubiquitination sites are in close proximity to Tyr118, suggesting potential interplay between phosphorylation and protein stability

• Functional significance of multiple modifications:

  • Tyr118 and Tyr31 phosphorylation together create binding sites for SH2 domain-containing proteins, particularly Crk, enhancing downstream signaling

  • Phosphorylation at Tyr118 may influence nearby ubiquitination sites, potentially affecting protein stability or localization

• Hierarchical relationships:

  • S85 phosphorylation by p38 MAPK (MAPK14) or CDK5 may influence subsequent Tyr118 phosphorylation

  • S272 phosphorylation by PAK1/PAK2/PAK4 occurs in a different domain but may allosterically affect tyrosine phosphorylation

Understanding these interrelationships is essential for comprehensive interpretation of paxillin regulation in complex cellular processes and for designing experiments that account for multiple modification states .

What is the role of paxillin Tyr118 phosphorylation in focal adhesion dynamics and cell migration?

Paxillin Tyr118 phosphorylation plays a central role in regulating focal adhesion dynamics and cell migration through several mechanisms:

• Focal adhesion formation and turnover:

  • Phosphorylation at Tyr118 creates binding sites for SH2 domain-containing proteins, particularly Crk

  • This recruitment initiates assembly of signaling complexes containing DOCK180 and Rac1, which regulate actin cytoskeleton remodeling essential for cell migration

  • The phosphorylation state influences adhesion stability, with dynamic phosphorylation/dephosphorylation cycles controlling adhesion turnover rates

• Integrin-mediated signaling:

  • Paxillin functions as a cytoskeletal protein involved in actin-membrane attachment at focal adhesions

  • Tyr118 phosphorylation occurs following integrin engagement with extracellular matrix components

  • This phosphorylation event represents a critical step in translating extracellular matrix cues into intracellular signaling events

• Cell migration regulation:

  • In epithelial cells, phosphorylation at Tyr118 is associated with enhanced migration during processes like epithelial-mesenchymal transition

  • In fibroblasts, phosphorylated paxillin localizes to focal contacts at the leading edge of migrating cells

  • The balance between phosphorylation at different sites (Tyr31, Tyr118) influences directional persistence and migration velocity

• Cancer cell invasion:

  • Elevated Tyr118 phosphorylation has been observed in invasive cancer cells

  • This modification contributes to enhanced migration and invasion capabilities through altered focal adhesion dynamics

  • Targeting kinases that phosphorylate paxillin at Tyr118 (FAK, Src) has emerged as a potential strategy to inhibit cancer cell invasion

• Mechanotransduction:

  • Phosphorylation at Tyr118 responds to mechanical forces applied to focal adhesions

  • This mechanism allows cells to sense and respond to changes in substrate stiffness and applied tension

Understanding these functions has significant implications for research in cell migration, cancer metastasis, wound healing, and development .

Why might I observe inconsistent results when detecting phospho-paxillin (Tyr118) in different experimental setups?

Inconsistent phospho-paxillin (Tyr118) detection can result from several factors that require systematic troubleshooting:

• Biological variability sources:

  • Cell density effects: Confluent cultures often show reduced phosphorylation due to contact inhibition

  • Passage number variation: Higher passage cells may have altered signaling networks

  • Stimulation timing: Phosphorylation at Tyr118 is dynamic and time-dependent following stimulation with growth factors or adhesion to ECM

• Technical variables impacting detection:

  Variable	Impact	Recommendation
  Sample handling time	Rapid dephosphorylation occurs	Process samples within 5 minutes of lysis
  Phosphatase inhibitor freshness	Degraded inhibitors allow dephosphorylation	Prepare fresh inhibitors for each experiment
  Antibody dilution	Too dilute fails to detect, too concentrated increases background	Optimize within manufacturer's range (1:500-1:2000)
  Blocking reagent	Milk contains phosphatases	Use BSA for blocking with phospho-specific antibodies
  Antibody lot variation	Epitope recognition differences between lots	Validate new lots against previous results

• Common causes of false negatives:

  • Ineffective phosphatase inhibition during sample preparation: Ensure complete inhibitor cocktail is used

  • Antibody blocked by sample buffer components: Check compatibility of buffer components

  • Degradation of phospho-epitope during extended storage: Process samples promptly and store at -20°C with glycerol as recommended by manufacturers

• Common causes of false positives:

  • Cross-reactivity with other phospho-tyrosine proteins: Verify with proper controls including peptide competition

  • Insufficient blocking: Follow recommended blocking protocols with BSA rather than milk

  • Contamination of phosphatase inhibitors in negative control samples: Prepare controls separately

• Standardization approaches:

  • Include standard positive control (e.g., A431 cells + EGF) in every experiment

  • Normalize phospho-signal to total paxillin within each sample

  • Consider using phosphatase treatment controls to establish baseline

Implementing these troubleshooting strategies based on manufacturer recommendations can significantly improve consistency in phospho-paxillin (Tyr118) detection across experiments .

How can I quantitatively analyze phospho-paxillin (Tyr118) levels in relation to total paxillin?

Accurate quantification of phospho-paxillin relative to total paxillin requires rigorous methodological approaches:

• Western blot quantification methods:

  • Sequential detection on same membrane: Strip and reprobe with total paxillin antibody

  • Parallel detection on duplicate membranes: Load identical samples on two gels

  • Two-color fluorescent detection: Use different species antibodies with distinct fluorophores

• Normalization strategies:

  • Direct ratio calculation: phospho-paxillin signal ÷ total paxillin signal

  • Fold change relative to control: normalized to both total paxillin and baseline condition

  • Verify signal is within linear detection range by running a dilution series of positive control

• Control samples for quantification:

  • Positive control: A431 cells treated with EGF show robust phosphorylation at Tyr118

  • Phosphatase-treated control: Establishes background signal level

  • Peptide competition control: Pre-incubation with phospho-peptide confirms signal specificity

• ELISA-based quantification options:

  • Several Phospho-PXN (Tyr118) antibodies are validated for ELISA applications

  • Typical dilutions range from 1:10000 for ELISA compared to 1:500-1:2000 for Western blot

  • Sandwich ELISA approach using capture by total paxillin antibody and detection with phospho-specific antibody provides most accurate quantification

• Data presentation and statistics:

  • Present both representative blot images and quantification from multiple independent experiments

  • Include error bars representing standard deviation or standard error

  • Apply appropriate statistical tests for comparing multiple conditions

  • Consider log transformation for ratio data before parametric statistical analysis

These quantitative approaches enable robust comparative analyses across experimental conditions and improve reproducibility of phosphorylation studies, as evidenced by multiple antibody validation studies .

What emerging technologies are enhancing paxillin phosphorylation research?

Several cutting-edge technologies are transforming how researchers study phospho-paxillin (Tyr118) dynamics and function:

• Advanced imaging approaches:

  • FRET-based biosensors for real-time visualization of paxillin phosphorylation in living cells

  • Super-resolution microscopy techniques allow visualization of phospho-paxillin distribution within focal adhesions at nanoscale resolution

  • Live-cell imaging combined with phospho-specific antibodies enables temporal studies of phosphorylation dynamics

• Proteomic innovations:

  • Phospho-proteomic profiling can identify paxillin phosphorylation in complex protein mixtures

  • Mass spectrometry approaches allow absolute quantification of phosphorylation stoichiometry

  • Proximity labeling methods identify phosphorylation-dependent protein-protein interactions

• Genetic engineering technologies:

  • CRISPR/Cas9 genome editing enables introduction of phospho-null mutations (Y118F) or phospho-mimetic mutations (Y118D/E) at endogenous loci

  • Knock-in of fluorescently tagged paxillin allows simultaneous visualization of localization and phosphorylation state

  • Optogenetic control of kinase activity permits spatial and temporal control of paxillin phosphorylation

• Computational approaches:

  • Molecular dynamics simulations predict structural changes induced by Tyr118 phosphorylation

  • Machine learning algorithms identify patterns in phosphorylation dynamics across experimental conditions

  • Systems biology modeling integrates paxillin phosphorylation into broader signaling networks

• Translational technologies:

  • Patient-derived organoids allow study of paxillin phosphorylation in disease-relevant contexts

  • Engineered biomaterials with defined mechanical properties for studying mechanosensitive phosphorylation

  • High-throughput screening platforms identify compounds that modulate paxillin phosphorylation

These technological advances enable unprecedented insights into the spatiotemporal dynamics and functional consequences of paxillin phosphorylation events, opening new avenues for understanding fundamental cell biology and disease mechanisms .

What are the implications of paxillin Tyr118 phosphorylation research for disease understanding and therapeutics?

Paxillin Tyr118 phosphorylation research has significant implications for understanding and potentially treating various diseases:

• Cancer research applications:

  • Altered paxillin phosphorylation patterns are observed in multiple cancer types

  • Phosphorylation at Tyr118 influences cancer cell migration, invasion, and metastasis

  • FAK and Src kinase inhibitors that indirectly target paxillin phosphorylation are in clinical development

  • Phospho-paxillin status may serve as a biomarker for predicting metastatic potential or therapeutic response

• Fibrotic disorders:

  • Dysregulated paxillin phosphorylation contributes to myofibroblast activation and excessive ECM production

  • Targeting the kinases responsible for Tyr118 phosphorylation may provide therapeutic avenues for fibrotic conditions

  • Understanding paxillin's role in mechanotransduction could inform treatments for fibrotic diseases

• Inflammatory and immune disorders:

  • Paxillin phosphorylation affects immune cell migration and adhesion

  • Modulation of Tyr118 phosphorylation could influence inflammatory cell recruitment and function

  • Immunological synapse formation may involve regulated paxillin phosphorylation

• Cardiovascular disease:

  • Endothelial cell function depends on properly regulated paxillin phosphorylation

  • Vascular smooth muscle cell migration during atherogenesis involves paxillin signaling

  • Cardiac remodeling after injury involves altered focal adhesion signaling including paxillin phosphorylation

• Neurodegenerative conditions:

  • Neuronal growth cone navigation relies on regulated paxillin phosphorylation

  • Axon regeneration after injury involves reactivation of developmental pathways including paxillin signaling

  • Glial cell responses to injury are mediated in part through adhesion-dependent signaling

These research directions highlight the importance of understanding paxillin Tyr118 phosphorylation beyond basic cell biology, with potential translational applications across multiple disease areas. The development of specific tools like Phospho-PXN (Tyr118) antibodies has been instrumental in advancing this field .