Phospho-PXN (Y118) Recombinant Monoclonal Antibody

What is PXN and why is Y118 phosphorylation significant?

Paxillin (PXN) is a cytoskeletal protein involved in actin-membrane attachment at sites of cell adhesion to the extracellular matrix, known as focal adhesions. It functions as a critical adaptor protein that recruits other proteins such as TRIM15 to focal adhesions . PXN consists of five LD domains at the N-terminus that participate in extensive protein-protein interactions and four LIM domains at the C-terminus involved in binding additional proteins .

Phosphorylation at tyrosine 118 (Y118) is particularly significant as it occurs during integrin-mediated cell adhesion, embryonic development, fibroblast transformation, and following stimulation by mitogens . This specific phosphorylation site has been associated with increased cell motility and invasion in cancer cells, suggesting its importance in cancer progression and metastasis . Y118 phosphorylation promotes the activation of RAC1 via CRK/CrKII, thereby enhancing migration and invasion processes .

What are the key differences between polyclonal and monoclonal Phospho-PXN (Y118) antibodies?

The fundamental difference between polyclonal and monoclonal Phospho-PXN (Y118) antibodies lies in their production methods and epitope recognition characteristics:

What controls should I include when using Phospho-PXN (Y118) antibodies?

Proper experimental controls are essential for validating antibody specificity and ensuring result reliability:

• Positive controls: Use cell lysates from cells treated with growth factors known to induce PXN phosphorylation, such as EGF (100ng/ml for 20 minutes) or Calyculin A (100nM for 60 minutes) . Western blot analysis has demonstrated that HeLa, A549, and HepG2 cell lines treated with these agents show increased phosphorylation at Y118 .

• Negative controls: Include untreated cell lysates from the same cell lines as your positive controls. Western blot data has confirmed that untreated HeLa, A549, and HepG2 cells show minimal phosphorylation at Y118 compared to treated samples .

• Dephosphorylation controls: Treat a portion of your positive control samples with phosphatase to remove phosphorylation and confirm antibody specificity for the phosphorylated form.

• Antibody validation: Consider using siRNA knockdown of PXN to verify antibody specificity, as demonstrated in studies showing PXN is required for focal adhesion formation in HeLa cells .

• Loading controls: Include antibodies against total PXN or housekeeping proteins to normalize phospho-signal and account for loading variations.

What are the recommended applications and dilutions for Phospho-PXN (Y118) antibodies?

Based on manufacturer recommendations and experimental validations, Phospho-PXN (Y118) antibodies can be used in several research applications with specific dilution ranges:

When working with new antibody lots or experimental systems, it is advisable to perform a dilution series to determine the optimal concentration that provides the best signal-to-noise ratio. The theoretical molecular weight of PXN is approximately 68 kDa, which should be used as a reference point when analyzing Western blot results .

How does PXN phase separation contribute to focal adhesion assembly?

Recent research has revealed that PXN undergoes liquid-liquid phase separation (LLPS), which plays a crucial role in focal adhesion assembly and dynamics. PXN consists of mostly disordered N-terminal regions, which contribute to its ability to undergo phase separation . This process is initiated by homotypic interactions but forms complex condensates when other client proteins are present under physiological conditions .

Experimentally, researchers have manipulated PXN phase separation using optogenetic tools to create de novo membrane-associated macromolecular assemblies. These synthetic structures efficiently concentrate cargo proteins belonging to the focal adhesion proteome and recapitulate key molecular events during focal adhesion formation and maturation . The material properties of PXN condensates are modulated by actomyosin contraction, highlighting the dynamic nature of these structures .

To study this phenomenon:

• Utilize optogenetic approaches: Fusion of PXN with light-sensitive domains (such as Cry2 PHR-mCherry) enables controlled induction of PXN condensation and observation of subsequent recruitment of focal adhesion components .

• Temporal analysis: Monitoring the recruitment of different focal adhesion proteins to PXN condensates over time (10-60 minutes) allows for the study of the molecular cascade occurring during focal adhesion assembly .

• Fractionation experiments: Plasma membrane fractionation can determine if PXN condensates are plasma membrane-associated, a critical feature of functional focal adhesions .

This phase separation behavior of PXN represents a new paradigm for understanding focal adhesion dynamics and may provide novel therapeutic targets for conditions involving dysregulated cell adhesion.

What techniques can be used to study the relationship between PXN Y118 phosphorylation and cancer progression?

Phosphorylation of PXN at Y118 has been implicated in cancer cell motility and invasion. Several techniques can be employed to investigate this relationship:

• Phospho-specific Western blotting: Use Phospho-PXN (Y118) antibodies to compare phosphorylation levels between normal and cancer cell lines or tissues. This approach has been validated with multiple cell lines including HeLa, A549, and HepG2 treated with EGF or Calyculin A .

• Immunohistochemistry/immunofluorescence: Assess the spatial distribution of phosphorylated PXN in tissue sections or cultured cells to correlate with invasive phenotypes. Recommended dilutions range from 1:50 to 1:100 for optimal staining .

• Phosphomimetic and phosphodeficient mutants: Generate Y118E (phosphomimetic) or Y118F (phosphodeficient) PXN mutants to study the functional consequences of phosphorylation in cell migration, invasion, and signaling assays.

• Co-immunoprecipitation studies: Investigate how Y118 phosphorylation affects PXN interaction with binding partners such as CRK/CrKII and their downstream effectors like RAC1 .

• Live-cell imaging: Combine phospho-specific antibodies with fluorescent tags to track the dynamics of PXN phosphorylation during cell migration in real-time.

• Kinase inhibition studies: Use specific inhibitors of kinases responsible for Y118 phosphorylation (such as Src family kinases) to establish causative relationships between signaling pathways, PXN phosphorylation, and cancer cell behavior.

• 3D invasion assays: Correlate PXN Y118 phosphorylation levels with invasive capacity in three-dimensional matrices to better mimic in vivo conditions.

How do different stimuli affect PXN Y118 phosphorylation patterns?

Various stimuli can induce PXN phosphorylation at Y118, making them useful for generating positive controls and studying signaling pathways:

The phosphorylation of PXN at Y118 occurs through multiple upstream kinases, including MAPK1/ERK2 and PTK6 . The specific pattern and duration of phosphorylation may vary depending on the stimulus and cell type, reflecting different signaling contexts and functional outcomes.

When designing experiments to study these patterns:

• Include appropriate time-course analyses to capture both rapid and sustained phosphorylation events.

• Consider the use of specific kinase inhibitors to dissect the relative contributions of different upstream pathways.

• Combine phospho-specific Western blotting with immunofluorescence to assess both quantitative changes and spatial distributions of phosphorylated PXN.

What are the challenges in detecting PXN Y118 phosphorylation in different subcellular fractions?

Detecting phosphorylated PXN in different subcellular fractions presents several technical challenges that researchers should address:

• Dynamic localization: PXN can localize to multiple subcellular compartments including the cytoplasm, cytoskeleton, cell junction, focal adhesion, and cell cortex . This dynamic distribution requires careful fractionation protocols to preserve phosphorylation status.

• Phosphatase activity: Rapid dephosphorylation during sample preparation can lead to false negatives. Including phosphatase inhibitors (such as sodium orthovanadate, sodium fluoride, and β-glycerophosphate) in all buffers is essential.

• Extraction efficiency: PXN associated with the cytoskeleton or focal adhesions may require specialized extraction buffers to solubilize effectively without disrupting phosphorylation.

• Antibody accessibility: In intact structures such as focal adhesions, epitope masking may occur due to protein-protein interactions, potentially reducing antibody binding efficiency.

• Signal-to-noise ratio: Phosphorylated PXN may represent a small fraction of total PXN in certain compartments, requiring sensitive detection methods and appropriate controls.

To address these challenges:

• Use plasma membrane fractionation experiments to confirm membrane association of PXN assemblies, as demonstrated in studies of optogenetically-induced PXN condensates .

• Employ both biochemical fractionation and immunofluorescence approaches to correlate localization data across methodologies.

• Consider phospho-enrichment techniques such as phospho-peptide immunoprecipitation prior to analysis of low-abundance fractions.

• Validate fractionation quality using established markers for different subcellular compartments.

How should I optimize Western blot conditions for Phospho-PXN (Y118) detection?

Optimizing Western blot conditions for Phospho-PXN (Y118) detection requires attention to several critical parameters:

• Sample preparation: Lyse cells directly in hot SDS-PAGE sample buffer containing phosphatase inhibitors to immediately inactivate phosphatases. For adherent cells, rapid lysis on the culture dish is preferable to prevent phosphorylation changes during harvesting.

• Protein loading: Load 20-50 μg of total protein per lane, adjusting based on the abundance of phosphorylated PXN in your samples. Higher loading may be required for samples with low phosphorylation levels.

• Gel percentage: Use 8-10% acrylamide gels to achieve optimal separation around the 68 kDa range where PXN migrates .

• Transfer conditions: Optimize transfer time and voltage for proteins in this molecular weight range. Semi-dry transfers at 15V for 30 minutes or wet transfers at 100V for 1 hour are typical starting points.

• Blocking conditions: Use 5% BSA in TBST rather than milk, as milk contains phosphatases that may reduce signal from phospho-epitopes.

• Antibody incubation: Dilute primary antibody in 5% BSA/TBST at 1:500-1:2000 . Incubate overnight at 4°C with gentle agitation to maximize specific binding.

• Washing: Perform thorough washing steps (4-5 times for 5 minutes each) with TBST to reduce background.

• Detection system: Use highly sensitive chemiluminescent or fluorescent detection systems, particularly for samples with low phosphorylation levels.

• Stripping and reprobing: When analyzing both phosphorylated and total PXN, consider running duplicate gels rather than stripping and reprobing, as stripping can lead to signal loss, especially for phospho-epitopes.

Examples of successful Western blot results include detection of phosphorylated PXN in HeLa cells treated with EGF (100ng/ml for 20 minutes), A549 cells treated with Calyculin A (100nM for 60 minutes), and HepG2 cells treated with EGF .

What factors affect the specificity of Phospho-PXN (Y118) antibody detection?

Several factors can influence the specificity of Phospho-PXN (Y118) antibody detection:

• Antibody source and type: Recombinant monoclonal antibodies like clone 1F11 typically offer higher specificity for the precise phosphorylation site compared to polyclonal antibodies . The immunogen sequence used to generate the antibody strongly influences specificity - antibodies raised against synthetic phosphopeptides corresponding to residues surrounding Y118 of human PXN show high specificity .

• Cross-reactivity with similar phospho-motifs: The amino acid sequence surrounding Y118 (HVYSF) is the immunogen for many anti-phospho-PXN antibodies . Antibodies may potentially cross-react with similar phosphorylated motifs in other proteins.

• Dephosphorylation during sample preparation: Inadequate phosphatase inhibition can lead to rapid loss of phosphorylation, reducing specific signal.

• Antibody validation: Proper validation using phosphatase treatment, Y118 mutants, or PXN-depleted samples is essential to confirm specificity.

• Species reactivity: While many Phospho-PXN (Y118) antibodies react with human samples, reactivity with other species varies by antibody. Some antibodies show reactivity with human, mouse, and rat samples , while others may have more limited species reactivity.

• Buffer composition: The presence of detergents, salts, or other additives in buffers can affect epitope accessibility and antibody binding.

To maximize specificity:

• Validate new antibody lots with positive and negative controls.

• Use the antibody within the applications validated by manufacturers (WB, ELISA, IHC) .

• Consider the species reactivity of the antibody (human, mouse, rat) when designing experiments .

• Include appropriate blocking agents to minimize non-specific binding.

How can I integrate Phospho-PXN (Y118) data with other focal adhesion protein analyses?

Integrating Phospho-PXN (Y118) data with analyses of other focal adhesion proteins provides a more comprehensive understanding of adhesion dynamics and signaling networks:

• Multiplex immunofluorescence: Simultaneously detect Phospho-PXN (Y118) alongside other focal adhesion proteins such as FAK, vinculin, or zyxin to analyze spatial relationships and co-localization patterns. This approach can reveal temporal recruitment of different proteins, as seen in studies showing early recruitment of certain components to PXN condensates followed by later recruitment of F-actin crosslinking proteins .

• Sequential Western blotting: Analyze multiple focal adhesion proteins from the same samples by either running multiple gels or carefully stripping and reprobing membranes. This allows correlation of phosphorylation levels across different components of the adhesion complex.

• Co-immunoprecipitation studies: Use Phospho-PXN (Y118) antibodies for immunoprecipitation followed by Western blotting for interacting partners to identify phosphorylation-dependent interactions.

• Correlation with functional outcomes: Combine Phospho-PXN (Y118) data with functional assays such as migration, adhesion strength measurements, or traction force microscopy to establish relationships between phosphorylation status and mechanical/functional properties of focal adhesions.

• Temporal analysis: Study the time-dependent changes in PXN phosphorylation alongside other focal adhesion components during adhesion formation, maturation, and disassembly. Research has shown that PXN condensates gradually recruit different focal adhesion proteins over time (10-60 minutes), reflecting the molecular cascade of focal adhesion assembly .

• Proteomics approaches: Use phospho-proteomics to identify additional phosphorylation sites on PXN and other focal adhesion proteins that may regulate their functions and interactions.

• Bioinformatics integration: Employ pathway analysis and protein-protein interaction databases to contextualize Phospho-PXN (Y118) data within broader signaling networks.

What are common issues in Phospho-PXN (Y118) antibody experiments and how can they be resolved?

Researchers commonly encounter several issues when working with Phospho-PXN (Y118) antibodies:

• Weak or absent signal in Western blots:

  • Ensure cells were appropriately stimulated (e.g., with EGF or Calyculin A)

  • Verify phosphatase inhibitors were included in all buffers

  • Try shorter harvesting times to minimize dephosphorylation

  • Increase antibody concentration or incubation time

  • Use more sensitive detection methods

  • Consider enriching phosphoproteins prior to Western blotting

• High background or non-specific bands:

  • Optimize blocking conditions (5% BSA is typically preferred over milk)

  • Increase washing duration and frequency

  • Further dilute primary and secondary antibodies

  • Use a more specific recombinant monoclonal antibody

  • Pre-absorb antibody with non-phosphorylated peptide if available

• Inconsistent results between experiments:

  • Standardize cell culture conditions and stimulation protocols

  • Maintain consistent sample preparation techniques

  • Use the same antibody lot when possible

  • Include positive controls (e.g., EGF-treated HeLa cells) in each experiment

  • Normalize phospho-signal to total PXN levels

• Poor immunofluorescence staining:

  • Optimize fixation method (paraformaldehyde typically preserves phospho-epitopes better than methanol)

  • Adjust permeabilization conditions to maintain epitope accessibility

  • Try antigen retrieval methods if necessary

  • Use tyramide signal amplification for low-abundance phospho-proteins

  • Consider using monoclonal antibodies for higher specificity in imaging applications

• Loss of reactivity over time:

  • Store antibodies according to manufacturer recommendations (-20°C for up to 1 year)

  • Avoid repeated freeze-thaw cycles

  • Aliquot antibodies upon receipt

  • Add stabilizing proteins such as BSA if not already present in the formulation

How should I validate a new lot of Phospho-PXN (Y118) antibody?

Proper validation of new antibody lots is essential for experimental reproducibility:

• Positive control testing: Test the new lot alongside the previous lot using established positive controls such as EGF-treated HeLa cells or Calyculin A-treated A549 cells . Compare signal intensity, specificity, and background levels.

• Dose-response analysis: Perform a dilution series (e.g., 1:500, 1:1000, 1:2000) to identify the optimal working concentration for the new lot .

• Phosphatase treatment control: Treat a portion of positive control lysates with lambda phosphatase to confirm that the signal is phosphorylation-dependent.

• Peptide competition: If available, perform peptide competition assays using phosphorylated and non-phosphorylated peptides corresponding to the Y118 region to confirm specificity.

• Knockout or knockdown validation: Test the antibody on samples from PXN knockout or knockdown cells to verify the absence of signal.

• Phospho-mutant analysis: If possible, test the antibody on cells expressing Y118F (phospho-deficient) mutant PXN to confirm specificity for the phosphorylated residue.

• Cross-application validation: If the antibody is intended for multiple applications (WB, IHC, ELISA), validate it in each application separately .

• Record lot-specific characteristics: Document the performance characteristics of each lot, including optimal dilutions, detection sensitivity, and any special considerations for future reference.

What emerging techniques could enhance Phospho-PXN (Y118) research?

Several cutting-edge techniques show promise for advancing Phospho-PXN (Y118) research:

• Super-resolution microscopy: Techniques such as STORM, PALM, or STED microscopy can provide nanoscale resolution of phosphorylated PXN within focal adhesions, revealing spatial organization beyond the diffraction limit of conventional microscopy.

• Live-cell phospho-sensors: Genetically encoded FRET-based biosensors for PXN phosphorylation would enable real-time monitoring of phosphorylation dynamics in living cells with high spatiotemporal resolution.

• Optogenetic control of phosphorylation: Expanding on the optogenetic approaches used to study PXN phase separation , developing tools to specifically control Y118 phosphorylation using light would allow precise temporal manipulation of this modification.

• Mass spectrometry-based phosphoproteomics: Advanced mass spectrometry techniques can quantify multiple phosphorylation sites on PXN simultaneously, revealing potential crosstalk between Y118 and other modifications.

• Single-molecule tracking: Techniques to track individual phosphorylated PXN molecules within living cells could reveal mobility, binding kinetics, and molecular interactions at unprecedented resolution.

• Cryo-electron microscopy: Structural studies of phosphorylated PXN within focal adhesion complexes could provide atomic-level insights into how Y118 phosphorylation affects protein conformation and interactions.

• CRISPR-based screening: Genome-wide or targeted CRISPR screens focused on regulators of PXN Y118 phosphorylation could identify new components of this signaling pathway.

• Patient-derived organoids: Studying PXN Y118 phosphorylation in three-dimensional patient-derived organoids could better recapitulate the in vivo signaling environment and provide more translational insights.

These emerging techniques could help address unresolved questions about how PXN Y118 phosphorylation contributes to focal adhesion dynamics, cell migration, and disease processes such as cancer metastasis.