PXN Recombinant Monoclonal Antibody

What is PXN and what cellular functions does it regulate?

Paxillin (PXN) is a multifunctional adaptor protein that serves as a critical component in focal adhesions, acting as a molecular scaffold for various signaling proteins. It plays essential roles in cell adhesion, migration, and cytoskeletal organization. The dysregulation of paxillin can have significant implications in diseases like cancer and neurological disorders . PXN belongs to the paxillin protein family and functions primarily at focal adhesions where it mediates signal transduction from the extracellular matrix to intracellular signaling pathways. As an adaptor protein, it coordinates multiple protein-protein interactions necessary for cell movement and tissue development.

How are PXN recombinant monoclonal antibodies produced?

The production of PXN recombinant monoclonal antibodies involves several sophisticated biotechnological processes:

• PXN antibody DNA sequences are cloned from immunoreactive rabbits

• A synthesized peptide derived from human paxillin is used as the immunogen

• The cloned PXN antibody genes are inserted into plasmid vectors

• These recombinant plasmid vectors are transfected into host cells

• Host cells express the antibody, which is then harvested

• The antibody undergoes affinity-chromatography purification

• Final testing confirms reactivity with human PXN protein through ELISA, IHC, and FC applications

This in vitro expression system ensures consistent antibody production with high specificity compared to traditional hybridoma techniques.

In which tissues is PXN expressed?

PXN expression has been documented in multiple tissues and cell types, which is important for experimental planning:

This widespread expression pattern makes PXN antibodies useful for studying various physiological and pathological processes .

What applications are PXN recombinant monoclonal antibodies validated for?

PXN recombinant monoclonal antibodies have been validated for multiple research applications:

Validation typically includes testing with known positive controls and negative samples to ensure specificity and high affinity .

How do I select the appropriate PXN recombinant antibody clone for my experiment?

Selection of the optimal PXN antibody requires consideration of several factors:

• Target epitope: Different clones recognize distinct epitopes. For example, clone RM256 targets the N-terminus of human Paxillin , while other antibodies may target C-terminal regions (amino acids 305-559) . Choose based on:

  • The accessibility of the epitope in your experimental conditions

  • Whether you need to detect specific PXN isoforms

  • The domain of interest for your research question

• Species reactivity: Available antibodies show varying cross-reactivity:

  • Some are human-specific

  • Others react with multiple species including bovine, chicken, hamster, human, mouse, and rat

  • Consider sequence homology if your species is not explicitly listed

• Application compatibility: Some clones perform better in specific applications:

  • PXC-10 clone excels in ICC and WB

  • RM256 clone shows excellent results in IHC, ICC, and WB

  • Some antibodies are validated for all major applications including IF and IP

• Host species: Consider the host species (mouse or rabbit ) to avoid cross-reactivity issues in multi-color immunostaining experiments.

What strategies can I employ to validate PXN antibody specificity for my experimental system?

Thorough validation is critical for ensuring reliable results with PXN antibodies:

• Positive and negative tissue controls:

  • Use tissues with known PXN expression (e.g., placenta, liver) as positive controls

  • Include tissues with minimal expression as negative controls

  • Verify observed staining patterns against literature-reported localization patterns

• Knockdown/knockout validation:

  • Employ siRNA knockdown or CRISPR-Cas9 knockout of PXN

  • Compare antibody reactivity between wild-type and KD/KO samples

  • Loss of signal in KD/KO samples confirms specificity

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Decreased or abolished signal indicates specificity

  • Some manufacturers offer blocking peptides for this purpose

• Multiple antibody comparison:

  • Test multiple PXN antibodies targeting different epitopes

  • Consistent staining patterns increase confidence in specificity

  • Discrepancies may indicate isoform-specific detection or non-specific binding

• Molecular weight verification:

  • Confirm the detected band in WB matches the expected molecular weight (64.5kDa)

  • Consider potential post-translational modifications that might alter migration

How can I optimize PXN antibody protocols for challenging applications?

Researchers often encounter challenges when using PXN antibodies in specific applications:

For Western Blotting:

• Sample preparation considerations:

  • Use phosphatase inhibitors to preserve phosphorylation states of PXN

  • Include protease inhibitors to prevent degradation

  • Optimize lysis buffer composition based on subcellular localization

• Protocol optimization:

  • Use recommended dilutions (1:1000-1:2000) as starting points

  • Optimize blocking conditions to reduce background

  • Consider extended transfer times for larger proteins

  • Test both reducing and non-reducing conditions

For Immunohistochemistry/Immunocytochemistry:

• Fixation methods:

  • Test different fixatives (PFA, methanol, acetone) to preserve epitope accessibility

  • Optimize fixation time to balance structural preservation with antigen availability

• Antigen retrieval:

  • Compare heat-induced epitope retrieval methods (citrate, EDTA buffers)

  • Adjust pH and buffer composition based on epitope characteristics

  • Consider enzyme-based retrieval for certain samples

• Signal amplification:

  • Employ tyramide signal amplification for low-abundance targets

  • Use appropriate detection systems (ABC, polymer-based) based on sensitivity requirements

What considerations are important when using PXN antibodies in co-localization studies?

Co-localization studies require special attention to several factors:

• Antibody compatibility:

  • Select primary antibodies from different host species to avoid cross-reactivity

  • If using same-species antibodies, employ sequential staining protocols with blocking steps

  • Consider directly conjugated antibodies to simplify protocols

• Microscopy optimization:

  • Use appropriate filter sets to minimize spectral overlap

  • Employ confocal or super-resolution microscopy for precise localization

  • Collect single-color controls to set acquisition parameters and thresholds

• Analysis approaches:

  • Utilize quantitative co-localization analysis (Pearson's coefficient, Manders' overlap)

  • Implement automated analysis workflows to reduce bias

  • Consider 3D co-localization for volumetric samples

• Biological relevance:

  • Design experiments to reveal functional relationships between PXN and other proteins

  • Include appropriate controls (e.g., proteins known to co-localize or not co-localize with PXN)

  • Interpret results in the context of known PXN interaction partners and cellular functions

How do different PXN isoforms affect antibody selection and experimental interpretation?

PXN exists in multiple isoforms, which can complicate antibody selection and data interpretation:

• Isoform recognition:

  • Determine which isoforms are recognized by your antibody based on the epitope location

  • C-terminal antibodies (e.g., targeting amino acids 305-559) may detect multiple isoforms

  • N-terminal antibodies like RM256 may have isoform-specific detection properties

• Isoform-specific expression:

  • Different tissues may express varying isoform patterns

  • Consider tissue-specific isoform expression when interpreting results

  • Verify which isoform is relevant for your research question

• Post-translational modifications:

  • PXN undergoes extensive phosphorylation that may affect antibody binding

  • Acetylation, ubiquitination, and other modifications can alter epitope accessibility

  • Some antibodies may be sensitive to specific modification states

• Experimental design considerations:

  • Use isoform-specific primers for qPCR validation studies

  • Consider employing multiple antibodies targeting different regions

  • Include appropriate positive controls expressing known isoforms

How can I troubleshoot inconsistent results when using PXN antibodies?

When encountering inconsistent results with PXN antibodies, consider the following:

• Antibody quality assessment:

  • Check antibody lot-to-lot variation

  • Verify storage conditions (avoid repeated freeze-thaw cycles)

  • Test antibody activity with a known positive control

  • Consider reconstitution methods for lyophilized antibodies

• Protocol standardization:

  • Standardize sample preparation procedures

  • Control incubation times and temperatures precisely

  • Use automated systems where possible to reduce variability

  • Document all protocol deviations

• Sample-specific considerations:

  • Account for tissue/cell-specific expression levels

  • Adjust protocols based on sample fixation/preservation method

  • Consider the impact of disease state on protein expression or localization

  • Modify extraction methods based on subcellular localization

• Technical optimizations:

  • Try different blocking agents to reduce background

  • Adjust antibody concentration based on signal intensity

  • Optimize detection systems for sensitivity and specificity

  • Consider signal amplification methods for low-abundance targets

What quality control measures ensure reliable results with PXN antibodies?

Implement these quality control measures to ensure experimental rigor:

• Positive and negative controls:

  • Include tissue/cells known to express or lack PXN

  • Use siRNA knockdown or CRISPR knockout samples as definitive controls

  • Compare staining patterns with published results

• Technical replicates:

  • Perform experiments in triplicate

  • Use multiple antibody lots when possible

  • Validate findings with complementary techniques (e.g., IF and WB)

• Validation of commercial claims:

  • Verify manufacturer's application claims in your specific system

  • Document any deviations from expected results

  • Consider independent validation if results contradict literature

• Documentation and standardization:

  • Record detailed experimental conditions

  • Standardize image acquisition parameters

  • Implement quantitative analysis methods to reduce subjective interpretation

  • Maintain comprehensive records of antibody performance across experiments

How can PXN antibodies be utilized in cancer research?

PXN antibodies offer valuable tools for cancer research applications:

• Diagnostic and prognostic markers:

  • Evaluate PXN expression in different cancer types

  • Correlate expression with clinical outcomes

  • Assess subcellular localization changes during tumor progression

• Metastasis studies:

  • Investigate PXN's role in cell migration and invasion

  • Examine focal adhesion dynamics in metastatic cells

  • Study PXN phosphorylation state as an indicator of invasive potential

• Therapeutic target validation:

  • Monitor PXN expression/modification in response to treatments

  • Evaluate effects of PXN knockdown on cancer cell behavior

  • Identify PXN-interacting proteins as potential drug targets

• Experimental approaches:

  • Implement tissue microarrays for high-throughput analysis

  • Combine with other markers to develop comprehensive profiles

  • Utilize multiplexed imaging to assess pathway activation

What are the emerging applications for studying PXN in neurological disorders?

The role of PXN in neurological disorders represents an emerging research area:

• Neurodevelopmental studies:

  • Investigate PXN's role in neuronal migration

  • Study axon guidance and synapse formation

  • Examine cytoskeletal remodeling during development

• Neurodegenerative disease research:

  • Assess PXN expression in models of neurodegeneration

  • Investigate its role in neuronal survival and repair

  • Study potential contributions to protein aggregation pathways

• Experimental considerations:

  • Use brain-derived cell lines or primary neuronal cultures

  • Implement sophisticated imaging to capture morphological changes

  • Combine with neuronal markers for co-localization studies

  • Consider the blood-brain barrier when designing in vivo experiments

How can researchers study PXN interactions with the cytoskeleton and signaling pathways?

To investigate PXN's role in cytoskeletal organization and signaling:

• Cytoskeletal interaction studies:

  • Use co-immunoprecipitation with PXN antibodies to identify binding partners

  • Perform proximity ligation assays to confirm direct interactions

  • Combine with actin or tubulin staining to visualize cytoskeletal associations

• Phosphorylation analysis:

  • Use phospho-specific antibodies alongside total PXN antibodies

  • Implement phosphatase treatments as controls

  • Correlate phosphorylation status with cellular behaviors

• Advanced techniques:

  • Apply live cell imaging to track PXN dynamics during cell migration

  • Implement FRET or BRET assays to study protein-protein interactions

  • Use optogenetic approaches to manipulate PXN function in real-time

• Experimental design considerations:

  • Control environmental factors affecting focal adhesion formation

  • Consider matrix composition and stiffness

  • Account for cell type-specific signaling contexts

What databases can I consult for information about PXN?

Researchers can access comprehensive PXN information through these databases:

These resources provide valuable information on protein structure, function, interactions, and disease associations that can inform experimental design and interpretation .

How should I cite PXN antibodies in my research publications?

Proper citation of antibodies is essential for research reproducibility:

• Essential information to include:

  • Manufacturer name and location

  • Catalog number

  • Clone designation (e.g., PXC-10, RM256)

  • Host species and antibody type (monoclonal/recombinant)

  • RRID (Research Resource Identifier) if available

• Sample citation formats:

  • "Anti-Paxillin Pxn Antibody (Monoclonal, PXC-10) (Boster Biological Technology, Pleasanton CA, USA, Catalog # MA1080)"

  • "Anti-Paxillin PXN Rabbit Monoclonal Antibody, Clone#RM256 (Boster Biological Technology, Pleasanton CA, USA, Catalog # M01033-1)"

• Methods section details:

  • Include dilution used

  • Detail any modifications to manufacturer's protocols

  • Describe validation performed

  • Note lot number if significant lot-to-lot variation exists