PXN Antibody, FITC conjugated

What is Paxillin (PXN) and why is it important in cellular research?

Paxillin is a 64 kDa cytoskeletal adapter protein that plays a crucial role in organizing and maintaining focal adhesions, which are essential structures for cell adhesion and migration. The biological significance of Paxillin extends to numerous physiological and pathological processes including embryogenesis, organogenesis, wound repair, inflammation, and cancer development .

At the molecular level, Paxillin contains specialized domains that facilitate protein-protein interactions, including:

• LD motifs

• LIM domains

• SH3 and SH2 binding domains

These structural elements serve as docking sites for cytoskeletal proteins, tyrosine kinases (including FAK, Pyk 2, Src), serine/threonine kinases, GTPase activating proteins, and other adapter proteins such as Actin, Vinculin, and Crk . This extensive interaction network positions Paxillin as a central hub in cellular signaling related to adhesion dynamics.

Paxillin expression has been documented across diverse tissues including:

What applications are most suitable for PXN Antibody, FITC conjugated?

FITC-conjugated PXN antibodies have been validated for multiple research applications, with varying dilution recommendations for optimal results :

The FITC conjugation offers distinct advantages, including direct visualization without secondary antibodies, which can simplify multi-labeling experimental designs. The excitation/emission profile (499/515 nm) is compatible with standard 488 nm laser lines available in most flow cytometers and fluorescence microscopes .

What are the recommended protocols for using PXN Antibody, FITC conjugated in immunofluorescence studies?

Successful immunofluorescence detection of Paxillin requires attention to several methodological details. Based on validated protocols, the following step-by-step approach is recommended:

For Paraffin-Embedded Tissue Sections:

• Perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• Block tissue sections with 10% goat serum to minimize non-specific binding

• Incubate with PXN antibody (2-5 μg/ml) overnight at 4°C

• Wash thoroughly with appropriate buffer

• For directly conjugated FITC-PXN antibodies, proceed directly to counterstaining

• Counterstain nuclei with DAPI

• Mount and analyze using appropriate filter sets (excitation ~499 nm, emission ~515 nm)

For Cultured Cells:

• Fix cells with 4% paraformaldehyde

• Permeabilize with appropriate buffer (critical for accessing intracellular Paxillin)

• Block with 10% normal goat serum

• Incubate with FITC-conjugated PXN antibody (optimal concentration determined experimentally)

• Counterstain nuclei with DAPI

• Mount and visualize using a fluorescence microscope with appropriate filters

For enzyme-based antigen retrieval, some protocols recommend IHC enzyme antigen retrieval reagent treatment for 15 minutes as an alternative to heat-mediated retrieval .

How should PXN Antibody, FITC conjugated be stored to maintain optimal activity?

Proper storage of FITC-conjugated PXN antibodies is essential for maintaining both antibody functionality and fluorescence intensity. The research literature recommends the following storage practices:

Most commercial preparations contain stabilizing components such as:

• 0.01M TBS or PBS (pH 7.4)

• 1% BSA (though BSA-free formulations are available upon request)

• 0.03% Proclin300 (preservative)

• 50% Glycerol (cryoprotectant)

It is important to note that some researchers may require BSA-free formulations for specific applications, which can often be specially prepared upon request from manufacturers .

What controls should be included when using PXN Antibody, FITC conjugated?

Rigorous experimental design requires appropriate controls to validate findings and ensure specificity. When using FITC-conjugated PXN antibodies, the following controls should be considered:

• Isotype Controls:

  • Include the appropriate isotype control antibody (e.g., rabbit IgG for rabbit-derived PXN antibodies)

  • Use at the same concentration as the experimental antibody

  • Helps distinguish specific staining from Fc receptor binding or other non-specific interactions

• Negative Controls:

  • Unstained samples (no primary or secondary antibody)

  • Primary antibody omission (secondary only)

  • These controls help establish background fluorescence levels

• Positive Controls:

  • Tissues or cell lines with known PXN expression (e.g., MDA-MB-231, A549, MCF-7 cells)

  • Helps validate the antibody's ability to detect the target

• Fluorescence Controls:

  • Single-color controls for compensation in multicolor flow cytometry

  • Autofluorescence controls, particularly important for tissues with high intrinsic fluorescence

• Specificity Validation:

  • Peptide competition assays may be used where the immunizing peptide blocks specific binding

  • Correlate staining pattern with known subcellular localization (cytoplasm, focal adhesions)

How can pH affect PXN antibody binding and what methodological adaptations should be considered?

The pH microenvironment can significantly impact antibody-antigen interactions, particularly in the context of tumor biology research. As demonstrated in several studies, pH-dependent effects warrant careful methodological consideration:

One study utilized pH-sensitive conjugates to exploit the acidic microenvironment of solid tumors, observing:

• Enhanced antibody recruitment at pH 6.0 compared to pH 7.4

• pH-dependent increase in fluorescence levels

• Significant changes in cellular binding properties based on pH conditions

These findings suggest several methodological implications for researchers:

• Buffer Optimization:

  • Buffer pH should be carefully controlled and reported

  • Effects of pH variation should be tested during protocol optimization

  • Consider pH as a variable in experimental design rather than a constant

• Cancer Research Applications:

  • The acidic microenvironment of tumors may alter antibody binding characteristics

  • pH-dependent conjugates might be leveraged for selective targeting

  • Comparison between normal and tumor tissue may require pH considerations

• Signal Interpretation:

  • Changes in signal intensity across pH ranges may reflect biological phenomena rather than technical issues

  • Quantitative analyses should account for potential pH effects

  • Validation across multiple pH conditions may strengthen findings

What strategies can optimize dual-labeling experiments using FITC-conjugated PXN antibodies?

Dual-labeling experiments present unique challenges that require careful optimization. For FITC-conjugated PXN antibodies, consider these advanced strategies:

• Spectral Compatibility Planning:

• Sequential Labeling Approach:

  • Complete PXN-FITC staining first

  • Block remaining binding sites with excess IgG

  • Proceed with second primary antibody from a different host species

  • Use spectrally distinct secondary antibody for second primary

• Signal Amplification Options:

  • For weak FITC signals, employ anti-FITC antibodies for signal amplification

  • Utilize biotin-streptavidin systems with non-FITC fluorophores

  • This approach is particularly useful when examining focal adhesions in thick tissue sections

• Anti-FITC Antibody Applications:

  • Anti-FITC antibodies can be useful in double-labeling experimental designs

  • They can amplify FITC signals when necessary

  • These antibodies help validate successful FITC conjugation

• Cross-Reactivity Mitigation:

  • Test antibodies individually before combining

  • Use highly cross-adsorbed secondary antibodies

  • Include appropriate blocking steps to minimize non-specific binding

How does fixation method affect epitope recognition by PXN antibody in different sample types?

Fixation methodology can significantly impact epitope accessibility and recognition by PXN antibodies. This consideration becomes particularly important when comparing results across different experimental systems:

• Paraformaldehyde Fixation Effects:

  • 4% paraformaldehyde is commonly used for cell fixation

  • PFA should be freshly prepared as long-term stored PFA forms formaldehyde polymers

  • PFA offers better tissue penetration compared to some alternatives

  • Overfixation may mask epitopes, while underfixation may compromise morphology

• Antigen Retrieval Requirements:

• Cell Type Considerations:

  • Different cell lines may require optimization of fixation conditions

  • Successful staining has been reported in diverse cell types including:

    • Epithelial: MDA-MB-231, A549, MCF-7, HeLa

    • Other: PC-3 (prostate cancer)

  • This suggests broad applicability across cell types but may require fine-tuning

• Enzyme-Based Alternatives:

  • Some protocols utilize enzyme antigen retrieval reagents (15 mins treatment)

  • This approach represents an alternative to heat-mediated retrieval

  • May be gentler for certain sensitive epitopes

• Correlation with Detection Method:

  • Flow cytometry typically requires robust permeabilization

  • Microscopy applications may need to balance permeabilization with morphological preservation

  • Western blotting requires complete protein denaturation

What technical considerations are important when analyzing PXN expression in cancer research using FITC-conjugated antibodies?

Cancer research presents unique challenges for PXN analysis due to complex microenvironments and altered protein expression patterns. Several technical considerations should be addressed:

• pH-Dependent Cancer Cell Targeting:
  Research has demonstrated that the acidic microenvironment of solid tumors can be exploited for selective antibody recruitment. When designing cancer-focused PXN studies:

  • Consider pH as an experimental variable (pH 6.0 vs. pH 7.4)

  • Evaluate potential pH-dependent changes in antibody binding

  • Design controls that account for microenvironmental differences

• Cancer-Specific Expression Analysis:
  PXN has been successfully detected in multiple cancer types including:

  • Breast cancer

  • Lung cancer

  • Thyroid papillary carcinoma

  • Laryngeal carcinoma

  • Hashimoto thyroiditis

• Functional Assessment Approaches:
  Beyond simple detection, advanced cancer research may examine functional aspects of PXN:

  • Cell lysis assays can assess antibody-dependent cellular cytotoxicity

  • Complement-dependent cytotoxicity studies

  • Correlation with migration and invasion phenotypes

• Technical Optimization Considerations:

  • FITC signal can be affected by the tumor microenvironment (pH, metabolites)

  • Autofluorescence is often elevated in cancer tissues

  • Signal-to-noise optimization may require additional blocking steps

  • Cross-validation with non-fluorescent detection methods is advisable

• Quantitative Analysis Parameters:

  • Flow cytometry can provide quantitative assessment of PXN expression levels

  • Consider using mean fluorescence intensity (MFI) rather than percent positive

  • Include multiple reference cell lines for comparative analysis

How can researchers troubleshoot common issues when using PXN antibody, FITC conjugated in flow cytometry?

Flow cytometry with FITC-conjugated PXN antibodies presents unique challenges requiring specific troubleshooting approaches:

• Weak Signal Intensity:

• High Background Problems:

  • Increase blocking stringency (use 10% normal serum)

  • Include proper controls (isotype control at same concentration)

  • Consider autofluorescence subtraction during analysis

  • Reduce antibody concentration if non-specific binding is observed

• Cell Preparation Optimization:

  • For intracellular staining, proper fixation is critical (4% paraformaldehyde recommended)

  • Use appropriate permeabilization buffer compatible with cytoskeletal proteins

  • Single-cell suspensions must be prepared carefully to preserve protein epitopes

  • Standardize cell numbers (e.g., 1μg antibody per 10^6 cells)

• Compensation Considerations:

  • FITC has potential spectral overlap with other fluorophores

  • Prepare single-color controls for compensation matrices

  • Consider alternative fluorophores if compensation is problematic

  • Use unstained controls to set basal fluorescence levels

• Validation Approaches:

  • Compare results with immunofluorescence microscopy

  • Verify subcellular localization pattern is consistent with known biology

  • Test multiple antibody clones if available

  • Consider parallel western blot analysis to confirm specificity

How can PXN antibodies contribute to understanding focal adhesion dynamics in cancer progression?

Paxillin serves as a critical component of focal adhesions, dynamic structures that mediate cell-extracellular matrix interactions and signaling. FITC-conjugated PXN antibodies offer valuable tools for investigating these processes:

• Metastasis Research Applications:

  • Focal adhesion turnover correlates with invasive potential

  • PXN phosphorylation status indicates activation of migration pathways

  • Quantitative analysis of focal adhesion size, number, and distribution provides insight into migration capacity

• Recommended Methodological Approaches:

  • Live-cell imaging with FITC-PXN for temporal dynamics

  • Co-staining with other focal adhesion proteins (vinculin, FAK)

  • Correlation of PXN localization with migration front in wound healing assays

  • Quantitative image analysis of focal adhesion morphometrics

• Interpretation Framework:

  • Increased peripheral PXN localization often indicates enhanced migration potential

  • Altered PXN phosphorylation may indicate aberrant signaling

  • Changes in focal adhesion size and turnover rate correlate with invasive capacity

  • Cytoskeletal coordination with focal adhesions reveals mechanotransduction mechanisms

What considerations are important when interpreting PXN antibody signals across different tissue types?

Tissue-specific characteristics can significantly influence both the technical aspects of staining and the biological interpretation of results:

• Tissue-Specific Expression Patterns:
  PXN expression varies considerably across tissues, requiring contextual interpretation:

  Tissue Type	Relative Expression	Localization Pattern	Functional Significance
  Brain/Testis	Variable	Often punctate	Specialized adhesions
  Cervix Carcinoma	Often elevated	Cytoplasmic & peripheral	Associated with invasion
  Fetal Kidney	Developmental pattern	Dynamic distribution	Morphogenesis role
  Leukemic T-cells	Context-dependent	Diffuse or clustered	Immune synapse function
  Liver	Generally moderate	Typically cell borders	Hepatocyte junctions
  Placenta	Relatively high	Complex pattern	Trophoblast invasion

• Technical Tissue-Specific Considerations:

  • Tissue autofluorescence varies dramatically (liver > kidney > brain)

  • Fixation requirements differ (brain tissue often requires gentler fixation)

  • Antigen retrieval efficacy is tissue-dependent

  • Background staining patterns vary by tissue type

• Interpretation Guidelines:

  • Always compare to appropriate tissue-matched controls

  • Consider developmental stage when examining embryonic tissues

  • Account for tissue-specific microenvironments (pH, matrix composition)

  • Correlate with known tissue architecture and cell type distribution

  • Validate findings across multiple detection methods when possible

How can PXN antibody, FITC conjugated be utilized in high-content screening approaches?

High-content screening offers powerful approaches for analyzing cellular phenotypes at scale. FITC-conjugated PXN antibodies can be integrated into these workflows:

• Assay Development Considerations:

  • Optimize fixation and staining protocols for automation compatibility

  • Determine appropriate cell seeding density for focal adhesion visualization

  • Establish robust image analysis pipelines for quantifying:

    • Focal adhesion number, size, and morphology

    • Cytoskeletal organization relative to focal adhesions

    • PXN phosphorylation status (using phospho-specific antibodies)

• Multiplexing Strategies:

  • Combine FITC-PXN with spectrally distinct markers:

    • Cytoskeletal markers (actin, tubulin)

    • Other focal adhesion proteins (vinculin, FAK)

    • Cell cycle or viability indicators

  • Design compensation strategies for automated imaging platforms

  • Consider sequential staining approaches for complex panels

• Quality Control Metrics:

  • Signal-to-background ratio optimization

  • Z'-factor calculation for assay robustness

  • Plate effects monitoring and normalization

  • Positive and negative control inclusion on each plate

  • Technical replicate consistency assessment

• Data Analysis Frameworks:

  • Machine learning approaches for phenotypic classification

  • Multiparametric analysis of focal adhesion characteristics

  • Correlation of PXN patterns with functional outcomes

  • Population heterogeneity assessment at single-cell resolution

What methodological approaches enable successful analysis of PXN in dynamic live-cell imaging?

While fixed-cell analysis with FITC-conjugated antibodies provides valuable snapshots, understanding PXN dynamics often requires live-cell approaches. Consider these methodological strategies:

• Complementary Approaches to Fixed-Cell Antibody Studies:

  • Generate PXN-GFP/YFP fusion proteins for live imaging

  • Validate fusion protein localization against antibody staining

  • Use photoconvertible or photoactivatable tags for pulse-chase experiments

  • Combine with fixed timepoint antibody analysis for validation

• Technical Optimization Parameters:

  • Minimize phototoxicity through:

    • Reduced exposure times

    • Increased camera sensitivity

    • Strategic temporal sampling

    • Antifade media formulations

  • Balance temporal resolution with experiment duration

  • Maintain physiological conditions (temperature, CO2, humidity)

• Analytical Frameworks:

  • Focal adhesion assembly/disassembly rate calculation

  • Directional persistence of migration correlation

  • Force transmission analysis using deformable substrates

  • Ratiometric analysis for protein recruitment sequences

  • Correlation of dynamics with cellular outcomes

• Integration with Fixed Antibody Data:

  • Use live-cell dynamic data to guide optimal fixation timepoints

  • Correlate live dynamics with antibody-based molecular state assessment

  • Validate key timepoints with comprehensive antibody panels

  • Develop integrated models incorporating both approaches

How should researchers approach validation of novel applications for PXN antibody, FITC conjugated?

Developing novel applications requires systematic validation approaches. When expanding the use of FITC-conjugated PXN antibodies to new experimental systems:

• Comprehensive Specificity Validation:

  • Genetic approaches:

    • siRNA/shRNA knockdown

    • CRISPR/Cas9 knockout

    • Overexpression systems

  • Biochemical approaches:

    • Peptide competition assays

    • Multiple antibody epitopes comparison

    • Correlation with mass spectrometry data

• Cross-Platform Validation:

  • Western blot confirmation of specificity

  • Immunoprecipitation to verify target interaction

  • Microscopy for localization pattern assessment

  • Flow cytometry for quantitative expression analysis

  • Tissue microarray analysis for expression patterns across multiple samples

• Experimental System Expansion:
  When applying to new experimental systems (cell types, tissues, or species):

  • Start with established positive controls

  • Systematically optimize key parameters (fixation, permeabilization, antibody concentration)

  • Document successful protocols in detail

  • Consider epitope conservation across species via sequence alignment

  • Validate with orthogonal detection methods

• Functional Validation:

  • Correlate antibody detection with known functional outputs

  • Assess antibody interference with protein function

  • Determine if epitope recognition is affected by post-translational modifications

  • Evaluate detection in physiologically relevant contexts