PXN (Ab-31) Antibody

What is the PXN (Ab-31) Antibody and what epitope does it recognize?

The PXN (Ab-31) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes the Paxillin protein. It was generated using a synthetic peptide sequence corresponding to amino acids 29-33 (T-P-Y-S-Y) of human Paxillin as the immunogen . This antibody detects endogenous levels of total Paxillin protein and is not phospho-specific unless otherwise indicated in specialized versions . The antibody recognizes Paxillin, a 68 kDa focal adhesion protein that plays a critical role in cell signaling and cytoskeletal organization .

What applications has the PXN (Ab-31) Antibody been validated for?

The PXN (Ab-31) Antibody has been validated for multiple laboratory applications:

The antibody has shown reproducible results across multiple cell lines, including 293 cells, A431, HeLa, MCF-7, and CACO-2, as well as in rat and mouse liver tissues .

How should the PXN (Ab-31) Antibody be stored to maintain its activity?

For optimal performance and stability, store the antibody at -20°C for long-term preservation . The antibody is typically supplied in a stabilizing solution containing phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, with 150mM NaCl, 0.02% sodium azide, and 50% glycerol . For short-term use (up to 6 months), the antibody can be stored at 4°C . Avoid repeated freeze-thaw cycles as these can compromise antibody activity and specificity . For convenience during regular use, consider preparing small working aliquots to minimize freeze-thaw cycles.

How should I optimize Western blot conditions when using PXN (Ab-31) Antibody for first-time detection?

When using the PXN (Ab-31) Antibody for Western blotting optimization, follow this systematic approach:

• Sample preparation: Prepare cell or tissue lysates using a buffer containing protease inhibitors. For detecting Paxillin, RIPA buffer has shown good results with 293, HeLa, MCF-7, and CACO-2 cell lines .

• Protein loading: Begin with 30 μg of total protein per lane, as validated in previous experiments .

• Gel selection: Use a 5-20% gradient SDS-PAGE gel running at 70V (stacking)/90V (resolving) for optimal separation .

• Transfer conditions: Transfer proteins to a nitrocellulose membrane at 150 mA for 50-90 minutes .

• Blocking: Block with 5% non-fat milk in TBS for 1.5 hours at room temperature .

• Primary antibody incubation: Start with a 1:500 dilution of PXN (Ab-31) Antibody in blocking buffer and incubate overnight at 4°C .

• Washing: Wash the membrane with TBS-0.1% Tween 3 times for 5 minutes each .

• Secondary antibody: Use a goat anti-rabbit IgG-HRP at 1:10,000 dilution for 1.5 hours at room temperature .

• Detection: Develop using an enhanced chemiluminescent (ECL) detection system .

• Optimization: If band intensity is suboptimal, adjust the primary antibody concentration within the recommended range (1:500-1:1000) or modify incubation times.

What controls should be included when using PXN (Ab-31) Antibody in immunohistochemistry experiments?

For rigorous immunohistochemistry experiments with PXN (Ab-31) Antibody, include the following controls:

• Positive tissue control: Use human cell lines or tissues known to express Paxillin, such as A431, HeLa, or MCF-7 cells .

• Negative tissue control: Include tissues known to have minimal or no Paxillin expression.

• Primary antibody omission control: Process sections without the primary antibody but with all other reagents to assess non-specific binding of the secondary antibody.

• Peptide competition control: Pre-incubate the antibody with excess immunizing peptide (the T-P-Y-S-Y sequence) to confirm specificity .

• Isotype control: Use normal rabbit IgG at the same concentration as the primary antibody to identify non-specific binding.

• Dilution series: Perform a titration of antibody dilutions (e.g., 1:25, 1:50, 1:100, 1:200) to determine optimal signal-to-noise ratio .

Start with a 1:50 dilution of the antibody for paraffin-embedded tissues and adjust based on signal strength and background levels .

How can I use PXN (Ab-31) Antibody to study focal adhesion dynamics in live cell imaging experiments?

For investigating focal adhesion dynamics using PXN (Ab-31) Antibody in live cell imaging:

• Antibody modification: Since the PXN (Ab-31) Antibody is not inherently fluorescent, consider using commercially available fluorophore-conjugated versions (FITC-conjugated) or secondary labeling strategies .

• Alternative approach: For live cell imaging, a more effective strategy is to:

  • Clone the Paxillin gene into a GFP or other fluorescent protein expression vector

  • Transfect cells with this construct

  • Use the PXN (Ab-31) Antibody in fixed samples as validation of the fluorescent protein localization

• Focal adhesion turnover analysis:

  • Fix cells at different time points after stimulation

  • Immunostain with PXN (Ab-31) Antibody at 1:50-1:100 dilution

  • Counterstain with appropriate markers for other focal adhesion components

  • Analyze colocalization and structural changes

• Co-visualization strategies: Combine PXN (Ab-31) staining with antibodies against other focal adhesion proteins such as FAK, vinculin, or integrin to study complex formation and dynamics .

• Data acquisition: Use confocal microscopy with appropriate filter sets to capture high-resolution images of focal adhesions at cell periphery.

What approaches can resolve conflicting data when PXN (Ab-31) Antibody shows unexpected molecular weight bands in Western blots?

When encountering unexpected bands in Western blots using PXN (Ab-31) Antibody:

• Analyze alternative Paxillin isoforms: The paxillin gene can be alternatively spliced to generate beta and gamma isoforms . Verify if the unexpected bands correspond to these known variants:

  • Main isoform: ~68 kDa

  • Alternative isoforms: may appear at different molecular weights

• Check for post-translational modifications:

  • Phosphorylation: Paxillin is heavily phosphorylated, particularly at tyrosine residues including Tyr31

  • Ubiquitination: May result in higher molecular weight bands

  • Proteolytic cleavage: Can produce lower molecular weight fragments

• Validate with knockdown/knockout samples:

  • Perform siRNA knockdown of Paxillin

  • Compare band patterns between control and knockdown samples

  • True Paxillin bands should be diminished in knockdown samples

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide sequence (T-P-Y-S-Y)

  • Specific bands should be eliminated or significantly reduced

• Cross-validation with other antibodies:

  • Use alternative Paxillin antibodies targeting different epitopes

  • Compare band patterns to identify consistent bands representing true Paxillin

• Sample preparation optimization:

  • Try different lysis buffers to ensure complete protein extraction

  • Include appropriate protease and phosphatase inhibitors

  • Vary denaturation conditions (temperature, time)

How can PXN (Ab-31) Antibody be utilized in studying Paxillin's role in cancer cell migration and invasion?

To investigate Paxillin's involvement in cancer cell migration and invasion using PXN (Ab-31) Antibody:

• Immunohistochemical analysis of tumor tissues:

  • Compare Paxillin expression between normal tissues, primary tumors, and metastatic sites

  • Use PXN (Ab-31) Antibody at 1:50-1:100 dilution on paraffin-embedded sections

  • Correlate expression patterns with clinical outcomes and invasion parameters

• Phosphorylation status assessment:

  • Use phospho-specific Paxillin antibodies alongside PXN (Ab-31) Antibody

  • Compare total Paxillin (detected by Ab-31) with phosphorylated forms

  • The ratio can indicate activation state of focal adhesions in migrating cells

• Cell migration assays:

  • Perform wound healing or Transwell migration assays

  • Fix cells at different time points and immunostain with PXN (Ab-31) Antibody

  • Analyze focal adhesion distribution at the leading edge versus cell body

• Co-immunoprecipitation studies:

  • Use PXN (Ab-31) Antibody to immunoprecipitate Paxillin complexes

  • Identify binding partners through Western blotting or mass spectrometry

  • Compare interactome between normal and cancer cells

• 3D culture systems:

  • Grow cancer cells in 3D matrices

  • Perform immunofluorescence with PXN (Ab-31) Antibody to visualize focal adhesions in a more physiologically relevant context

  • Compare with 2D cultures to identify invasion-specific changes in Paxillin localization and expression

What strategies can resolve high background issues when using PXN (Ab-31) Antibody in immunohistochemistry?

To reduce background and improve signal-to-noise ratio in immunohistochemistry with PXN (Ab-31) Antibody:

• Optimize antibody concentration:

  • Test a dilution series from 1:25 to 1:200, starting with the recommended 1:50-1:100 range

  • Select the highest dilution that still gives specific signal

• Improve blocking conditions:

  • Extend blocking time to 1-2 hours

  • Try different blocking agents (5% BSA, 5-10% normal goat serum, commercial blocking solutions)

  • Add 0.1-0.3% Triton X-100 for better penetration in tissue sections

• Optimize antigen retrieval:

  • Test different methods (heat-induced epitope retrieval with citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Adjust retrieval time (10-20 minutes)

• Modify washing procedures:

  • Increase washing times and number of washes (5 washes of 5 minutes each)

  • Add 0.05-0.1% Tween-20 to wash buffers

• Reduce non-specific binding:

  • Add 5% normal serum from the species of the secondary antibody to the primary antibody dilution

  • Pre-absorb the secondary antibody with tissue powder

• Tissue processing considerations:

  • Ensure tissues are properly fixed (overfixation can increase background)

  • Use freshly cut sections and minimize storage time

• Secondary antibody optimization:

  • Try different detection systems (HRP-polymer vs. avidin-biotin complex)

  • Reduce secondary antibody concentration

How can I validate the specificity of PXN (Ab-31) Antibody for my particular experimental system?

To comprehensively validate PXN (Ab-31) Antibody specificity in your experimental system:

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunogenic peptide (T-P-Y-S-Y sequence)

  • Run parallel assays with blocked and unblocked antibody

  • Specific signal should be eliminated or significantly reduced in the blocked condition

• Genetic validation:

  • Use Paxillin knockout or knockdown models (CRISPR-Cas9, siRNA, shRNA)

  • Compare antibody reactivity between wild-type and knockout/knockdown samples

  • Specific signal should be absent or reduced in knockout/knockdown samples

• Cross-species reactivity testing:

  • Test the antibody on samples from different species if your research involves multiple models

  • The antibody has documented reactivity with human samples, but validation may be needed for other species

• Multiple detection methods:

  • Compare results across different techniques (WB, IHC, ICC)

  • Consistent detection across methods supports specificity

• Multiple antibody validation:

  • Use alternative antibodies targeting different Paxillin epitopes

  • Concordant results with multiple antibodies support specificity

• Recombinant protein testing:

  • Test against purified recombinant Paxillin protein

  • Compare with lysates to confirm molecular weight

• Correlate with mRNA expression:

  • Compare protein detection patterns with Paxillin mRNA expression (qPCR, RNA-seq)

  • Similar expression patterns support antibody specificity

How can I integrate PXN (Ab-31) Antibody data with phosphoproteomics to analyze focal adhesion signaling networks?

For integrating PXN (Ab-31) Antibody data with phosphoproteomics:

• Experimental design for complementary analyses:

  • Use PXN (Ab-31) Antibody to determine total Paxillin levels via Western blot (1:500-1:1000 dilution)

  • In parallel, perform phosphoproteomic analysis of the same samples

  • Complement with phospho-specific Paxillin antibodies targeting key sites (Tyr31, Tyr118)

• Quantitative correlation analysis:

  • Normalize phosphopeptide intensities to total Paxillin levels detected by PXN (Ab-31) Antibody

  • Calculate phosphorylation stoichiometry for individual sites

  • Correlate changes across experimental conditions or time points

• Pathway integration:

  • Map Paxillin and its interactors in the focal adhesion kinase (FAK) pathway

  • Overlay phosphorylation data on pathway maps

  • Identify coordinated regulation of multiple components

• Co-immunoprecipitation with phospho-enrichment:

  • Use PXN (Ab-31) Antibody for immunoprecipitation of Paxillin complexes

  • Perform phosphopeptide enrichment on the immunoprecipitated material

  • Identify phosphorylation sites on Paxillin and its binding partners

• Dynamic analysis of stimulation responses:

  • Track total Paxillin (via PXN Ab-31) and site-specific phosphorylation after stimulation

  • Create temporal profiles of phosphorylation events

  • Determine sequence of phosphorylation in signaling cascades

• Data visualization and integration:

  • Create network visualizations incorporating total protein abundance and phosphorylation data

  • Use correlation heatmaps to identify co-regulated phosphorylation events

  • Implement mathematical modeling to predict phosphorylation dynamics

What are the methodological considerations when using PXN (Ab-31) Antibody for quantitative analysis of Paxillin expression across tissue types?

For rigorous quantitative analysis of Paxillin expression across tissues using PXN (Ab-31) Antibody:

• Sample preparation standardization:

  • Use consistent fixation protocols across all tissue types

  • Process all samples simultaneously when possible

  • Include multiple biological replicates (minimum n=3) per tissue type

• Quantification method selection:

  • For Western blot: Use densitometry with appropriate normalization controls

  • For IHC: Implement digital image analysis with standardized algorithms

  • Score both staining intensity and percentage of positive cells

• Normalization strategy:

  • Western blot: Normalize to stable housekeeping proteins (β-actin, GAPDH, tubulin)

  • Include loading controls on each gel/blot

  • For IHC: Use tissue microarrays when possible to ensure consistent staining conditions

• Dynamic range considerations:

  • Ensure antibody dilutions allow detection within the linear range

  • For Western blot, test multiple exposure times to avoid saturation

  • For IHC, use standardized DAB development times

• Statistical analysis approach:

  • Use appropriate statistical tests for multiple tissue comparisons (ANOVA with post-hoc tests)

  • Implement non-parametric tests if data does not follow normal distribution

  • Account for multiple testing when analyzing large tissue panels

• Technical validation:

  • Include technical replicates to assess method reproducibility

  • Assess intra- and inter-observer variability for IHC scoring

  • Calculate coefficient of variation for quantitative measurements

• Cross-platform validation:

  • Validate key findings using orthogonal methods (e.g., mass spectrometry)

  • Correlate protein expression with mRNA levels from the same tissues

  • Consider absolute quantification using recombinant protein standards