PANX1 Recombinant Monoclonal Antibody

What is Pannexin 1 (PANX1) and why is it important in research?

Pannexin 1 is a channel-forming glycoprotein that functions as a structural component of gap junctions and hemichannels involved in ATP release and nucleotide permeation. In humans, the canonical protein has 426 amino acid residues with a molecular mass of approximately 48.1 kDa. PANX1 is widely expressed across various tissue types and localizes primarily in the endoplasmic reticulum and cell membrane . Its importance in research stems from involvement in diverse physiological functions including blood pressure regulation, apoptotic cell clearance, oogenesis, and immune response regulation . Alternative splicing yields two different isoforms, and post-translational modifications, particularly N-glycosylation, are critical for its function and trafficking .

How do I select the appropriate PANX1 monoclonal antibody for my research?

Selection should be based on your specific application requirements and target epitopes. Consider these methodological steps:

• Epitope targeting: Different epitopes yield different subcellular localization patterns. For example, antibodies targeting the N-terminus (e.g., Mo503) highlight neuronal processes more than cell bodies, while those targeting the intracellular loop or C-terminus primarily label cell bodies .

• Application compatibility: Verify compatibility with your intended applications (WB, IHC, IF, ELISA). For instance, while many antibodies work well for Western blotting, only some are validated for immunohistochemistry in paraffin-embedded tissues .

• Species reactivity: Confirm the antibody's reactivity with your species of interest. Gene orthologs have been identified in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken species .

• Validation evidence: Review available validation data, particularly regarding specificity shown through knockout/knockdown models .

What control samples should I use to validate PANX1 antibody specificity?

A comprehensive validation approach should include:

• Positive controls: Use cell lines known to express PANX1 (e.g., MDCK cells) or tissue with high PANX1 expression (brain regions, macrophages) .

• Negative controls:

  • Parental cell lines without PANX1 expression (e.g., untransfected HeLa cells)

  • Primary antibody omission controls

  • PANX1 knockout tissue (note that some knockout models are hypomorphs rather than true knockouts)

  • PANX1 knockdown cells using siRNA or shRNA

• Tagged protein validation: In transfected systems, use antibodies against epitope tags (e.g., myc) to confirm co-localization with your PANX1 antibody .

How do I optimize Western blot protocols for PANX1 detection?

PANX1 detection by Western blot requires specific considerations due to its glycosylation pattern:

• Sample preparation:

  • For membrane proteins like PANX1, optimize lysis buffers containing appropriate detergents

  • Include protease inhibitors to prevent degradation

• Band interpretation:

  • Expect multiple bands (typically three) corresponding to different glycosylation states:

    • GLY0: Non-glycosylated (~36-42 kDa)

    • GLY1: Partially glycosylated (~43-48 kDa)

    • GLY2: Fully glycosylated (~48-52 kDa)

• Optimization steps:

  • If bands appear at unexpected molecular weights, consider:

    • Validating with knockout/knockdown controls

    • Blocking with specific peptides

    • Comparing multiple antibodies recognizing different epitopes

• Resolution considerations:

  • Use gradient gels (4-12% or 4-20%) for better separation of the different glycosylated forms

  • Longer running times may be necessary to clearly separate closely migrating bands

What are the optimal conditions for immunofluorescence with PANX1 antibodies?

For successful immunofluorescence experiments:

• Fixation protocol:

  • Paraformaldehyde (4%) fixation is commonly used for PANX1 detection

  • For neuronal tissues, perfusion fixation may yield better results than post-fixation

• Permeabilization:

  • Optimize detergent concentration and exposure time (Triton X-100 at 0.1-0.3%)

  • For membrane proteins like PANX1, excessive permeabilization may disrupt epitope integrity

• Antibody dilution:

  • Titrate antibody concentrations; common dilutions range from 1:100 to 1:500

  • Include proper negative controls at the same concentration

• Epitope consideration:

  • Different antibodies may reveal different subcellular localization patterns

  • For complete cellular distribution, consider using multiple antibodies targeting different domains

• Co-localization studies:

  • Use additional markers for subcellular compartments to confirm localization patterns

  • For cell membrane localization, GLY2 (fully glycosylated) PANX1 is the relevant form

How can I quantify PANX1 expression levels in different experimental conditions?

Depending on your research question, consider these quantification approaches:

• Western blot quantification:

  • Normalize PANX1 band intensities to appropriate loading controls

  • For complete assessment, quantify all three glycosylation states separately

  • Consider the ratio between different glycosylation states, as this may change under experimental conditions

• RT-qPCR analysis:

  • Useful for transcriptional regulation studies

  • Design primers spanning exon-exon junctions to avoid genomic DNA amplification

  • Note that PANX1 protein levels may be regulated post-transcriptionally, so mRNA levels may not directly correlate with protein abundance

• Flow cytometry:

  • Useful for cell surface expression analysis

  • Utilize non-permeabilized cells to detect only membrane-localized PANX1

  • Can be combined with other markers for cell-type specific analysis

• Imaging quantification strategies:

  • For immunofluorescence: measure integrated density of signal, corrected for background

  • For tissue sections: quantify signal intensity across different regions using unbiased automated approaches

How do I design experiments to study PANX1 channel activity in conjunction with antibody labeling?

Combining functional and structural studies requires careful experimental design:

• Dye uptake assays:

  • Use channel-permeable dyes (e.g., YO-PRO-1, ethidium bromide) to assess PANX1 channel opening

  • Correlate dye uptake with antibody labeling in fixed cells post-experiment

  • Include PANX1 channel blockers (carbenoxolone, probenecid, spironolactone) as controls

• ATP release measurements:

  • Quantify extracellular ATP as a functional readout of PANX1 activity

  • Stimulate cells using KCl to activate PANX1 channels

  • Compare results between control and PANX1-deficient samples

• Calcium imaging:

  • Since PANX1 may function as a Ca²⁺-leak channel, monitor intracellular Ca²⁺ dynamics

  • Correlate channel activity with localization patterns revealed by antibody labeling

• Patch-clamp electrophysiology:

  • Record PANX1 channel currents in combination with antibody labeling

  • Consider using antibodies non-disruptively (e.g., fluorescently labeled Fab fragments)

How can I investigate PANX1 protein-protein interactions using monoclonal antibodies?

Several approaches can be employed:

• Co-immunoprecipitation (Co-IP):

  • Use anti-PANX1 antibodies to pull down protein complexes

  • Verify interactions by Western blot for suspected binding partners

  • Example: PANX1 interaction with β-catenin has been demonstrated using Co-IP in melanoma cells

• Proximity Ligation Assay (PLA):

  • Detect protein interactions within 40 nm distance

  • Requires two primary antibodies from different species

  • Successfully applied to detect PANX1-stomatin interaction in red blood cells (27.09% PLA+ cells)

  • Control validation showed significantly lower signals (1.57-6.13% PLA+) in negative controls

• Immunofluorescence co-localization:

  • Analyze pixel overlap between PANX1 and potential interaction partners

  • Calculate Pearson's or Mander's coefficients for quantitative assessment

  • Use super-resolution microscopy for enhanced spatial resolution

• FRET/BRET assays:

  • For dynamic interaction studies in living cells

  • May require protein tagging, but antibodies can be used for validation

What approaches can I use to study PANX1 in tissues with low expression levels?

For challenging detection scenarios:

• Signal amplification methods:

  • Tyramide signal amplification (TSA) can increase detection sensitivity

  • Multiplex immunofluorescence with spectral unmixing to separate weak signals from background

  • Consider RNAscope for combined mRNA/protein detection

• Enrichment strategies:

  • Membrane fractionation to concentrate PANX1 protein

  • Immunoprecipitation followed by Western blot for increased sensitivity

  • Consider stimulating cells to upregulate PANX1 expression (e.g., TLR agonists in macrophages)

• Alternative detection systems:

  • Highly sensitive detection systems like biotin-streptavidin amplification

  • Quantum dot-conjugated secondary antibodies for improved signal-to-noise ratio

  • Digital droplet PCR for extremely low abundance transcript detection

• Tissue-specific considerations:

  • Focus on regions with higher expression (e.g., in brain: cerebellum, hippocampus, olfactory bulb, and thalamus)

  • Use wide-field mosaic confocal microscopy for large area imaging while maintaining resolution

How do I resolve contradictory results from different PANX1 antibodies?

When faced with discrepancies:

• Systematic comparison approach:

  • Test multiple antibodies targeting different epitopes in parallel

  • Include appropriate positive and negative controls for each antibody

  • Document exact experimental conditions for reproducibility

• Epitope accessibility considerations:

  • Different fixation methods may affect epitope exposure

  • Post-translational modifications may mask certain epitopes

  • Protein conformation or interactions may block antibody binding sites

• Validation hierarchy:

  • Prioritize results from antibodies validated with knockout/knockdown controls

  • Consider findings from orthogonal methods (e.g., mass spectrometry)

  • Examine antibody performance across multiple applications

• Interpretation framework:

  • Different antibodies may reveal different aspects of PANX1 biology

  • For example, the Mo503 antibody labels neuronal processes more than cell bodies, while other antibodies show stronger cell body labeling

  • These differences may reflect genuine biological variation in protein conformation or modification

What are the key considerations when using PANX1 antibodies in different species?

For cross-species applications:

• Epitope conservation analysis:

  • Align sequences of the antibody's epitope region across target species

  • Higher conservation increases likelihood of cross-reactivity

  • Gene orthologs of PANX1 have been identified in mouse, rat, bovine, frog, zebrafish, chimpanzee and chicken

• Validation requirements:

  • Perform species-specific validation even if cross-reactivity is claimed

  • Use tissue from knockout animals of the target species when available

  • Verify that banding patterns match expected molecular weights for that species

• Tissue-specific optimization:

  • Adjust protocols for species-specific tissue architecture

  • Optimize fixation times based on tissue density and composition

  • Consider antigen retrieval modifications for different species

• Common pitfalls:

  • Evolutionary differences in post-translational modifications

  • Species variation in splice variants

  • Different expression patterns across homologous tissues

How can I distinguish between different PANX1 isoforms or post-translationally modified forms?

For detailed analysis of PANX1 variants:

• Glycosylation analysis:

  • Treat samples with glycosidases (PNGase F, Endo H) to remove N-linked glycans

  • Compare migration patterns before and after treatment

  • The three glycosylation states (GLY0, GLY1, GLY2) can be resolved on Western blots

• Isoform-specific detection:

  • Use antibodies targeting regions unique to specific splice variants

  • Design PCR primers to distinguish between splice variants at the mRNA level

  • Consider mass spectrometry for unambiguous isoform identification

• Phosphorylation status:

  • Use phospho-specific antibodies if available

  • Employ phosphatase treatments as controls

  • Consider Phos-tag gels for mobility shift detection of phosphorylated forms

• Subcellular distribution correlation:

  • Fully glycosylated PANX1 (GLY2) localizes predominantly to the plasma membrane

  • GLY0 and GLY1 species are typically found in intracellular compartments

  • Use this information to interpret localization patterns observed with different antibodies

How can I use PANX1 antibodies in conjunction with advanced imaging techniques?

Leverage cutting-edge imaging approaches:

• Super-resolution microscopy:

  • STORM/PALM techniques can resolve PANX1 channel distribution below diffraction limit

  • SIM or STED microscopy for improved visualization of subcellular localization

  • Antibody selection is crucial - smaller probes (Fab fragments) may provide better resolution

• Live cell imaging considerations:

  • Consider membrane-impermeant antibodies for surface-exclusive labeling

  • Use minimally disruptive labeling techniques that don't interfere with channel function

  • Validate that antibody binding doesn't alter channel properties

• Correlative light and electron microscopy (CLEM):

  • Combine immunofluorescence with ultrastructural analysis

  • Requires careful sample preparation and specialized probes

  • Provides nanometer-scale context for PANX1 localization

• Large-scale tissue imaging:

  • Wide field mosaic confocal microscopy allows imaging large regions while maintaining resolution

  • Automated acquisition and stitching techniques permit whole-brain section analysis

  • Used successfully to map PANX1 distribution across brain regions

What are the methodological considerations for studying PANX1 in specific disease models?

Disease-specific approaches include:

• Cancer research applications:

  • PANX1 interacts with β-catenin in melanoma cells to modulate growth and metabolism

  • For melanoma studies, consider co-immunoprecipitation to assess PANX1-β-catenin interaction

  • Quantify effects of PANX1 knockdown on β-catenin protein levels and localization

• Inflammatory conditions:

  • In macrophages, PANX1 expression is upregulated by diverse stimuli promoting pyroptosis

  • TLR agonists (e.g., R848) or bacterial bioparticles can stimulate PANX1 expression

  • Monitor both mRNA and protein levels as they may be differentially regulated

• Neurological disorders:

  • Study PANX1 in brain regions relevant to specific conditions

  • Cerebellum, hippocampus, olfactory bulb, and thalamus show high PANX1 expression

  • Consider cell-type specific expression patterns (neurons vs. glia)

• Hematological research:

  • PANX1 drives ATP release from red blood cells under stress conditions

  • KCl stimulation can be used to activate PANX1 channels

  • PANX1 inhibitors (carbenoxolone, probenecid, spironolactone) can serve as controls

How can I develop multiplex assays incorporating PANX1 antibodies?

For comprehensive analysis:

• Multiplexed immunofluorescence strategies:

  • Use primary antibodies from different species

  • Employ directly conjugated primary antibodies with non-overlapping fluorophores

  • Consider sequential detection with antibody stripping between rounds

• Flow cytometry applications:

  • Combine surface PANX1 detection with cell type-specific markers

  • Add functional readouts (e.g., calcium indicators, viability dyes)

  • Carefully titrate antibodies to minimize spectral overlap

• Single-cell analysis integration:

  • Combine PANX1 protein detection with transcriptomic analysis

  • Consider CITE-seq approaches for simultaneous protein and RNA profiling

  • Validate findings using spatial transcriptomics with protein co-detection

• High-content screening applications:

  • Develop assays measuring PANX1 levels, localization, and functional readouts

  • Optimize for automated image acquisition and analysis

  • Include appropriate controls for normalization and quality control