PANX1 Antibody

What is PANX1 and why is it important in neuroscience research?

PANX1 (Pannexin 1) is a channel protein highly expressed in the central nervous system that releases cytosolic ATP in response to signaling pathways. It plays critical roles in several neuronal processes, making it an important research target. PANX1 contributes to "ATP-induced ATP release" mechanisms whereby ATP stimulation of P2X or P2Y receptors signals the opening of Panx1 channels . This channel is involved in:

• Immune response activation in neurons

• NMDA receptor epileptiform electrical activity in the hippocampus

• Formation of the neuronal inflammasome

• Seizure activity (releasing ATP when induced by kainic acid)

• Paracrine wave signaling allowing cells to respond to stresses

PANX1 is widely expressed across various brain regions including the cerebellum, hippocampus, cortex, and olfactory bulb, making it essential for understanding brain function and pathology .

What are the main applications of PANX1 antibodies in research?

PANX1 antibodies serve multiple crucial functions in research:

Additionally, these antibodies are invaluable for comparing expression between wild-type and knockout models, studying expression patterns across brain regions, and investigating PANX1 levels in disease models .

What is the molecular weight of PANX1 and what band patterns should I expect in Western blots?

Although PANX1 has a calculated molecular weight of approximately 48 kDa (for the 426 amino acid human protein), researchers should expect several band patterns in Western blots due to post-translational modifications:

• Main PANX1 bands appear in the 36-55 kDa region

• Glycosylated forms typically appear at ~50 kDa

• Dimers or multimeric forms have been reported at ~90 kDa

• Some antibodies detect bands at ~20 kDa that decrease in knockout tissues

The observed molecular weight varies depending on:

• The specific antibody used

• Tissue or cell type analyzed

• Sample preparation conditions

• Species differences

Commercial antibodies report observed molecular weights of 45-50 kDa and 90 kDa, with the larger band likely corresponding to dimeric forms . Validation with knockout tissues is highly recommended due to potential non-specific bands.

How do I validate the specificity of a PANX1 antibody?

Validating PANX1 antibody specificity requires multiple complementary approaches:

• Peptide competition/blocking experiments:

  • Pre-incubate the antibody with its immunizing peptide

  • Use in Western blots and immunohistochemistry

  • Look for dose-dependent reduction of signal with increasing peptide concentration

• Knockout model validation:

  • Compare patterns between wild-type and PANX1 knockout tissues

  • Expect reduction/elimination of bands in the 36-55 kDa region

  • Note that some antibodies may show residual bands in knockout tissues

• Recombinant expression systems:

  • Test on cells transfected with tagged PANX1 (e.g., myc-tagged)

  • Confirm co-localization with antibodies against the tag

  • Include non-transfected cell lines as negative controls

• Multiple antibody comparison:

  • Use antibodies targeting different PANX1 epitopes

  • Compare labeling patterns across identical samples

  • Consistent patterns increase confidence in specificity

Even "good" antibodies may show tissue-dependent variability and some non-specific bands. As noted in the literature, "while Panx1 labeling with recombinant Panx1 in cultured cells gave consistent results, the Western blots from KO animals were not entirely clean or consistent between tissues and/or KO mice" .

What are the differences between various anti-PANX1 antibodies used in research?

Different anti-PANX1 antibodies target distinct epitopes across the protein, leading to important performance variations:

These differences reflect:

• Unique or differentially accessible PANX1 populations

• Variations in antibody specificity

• Potential post-translational modifications affecting epitope accessibility

Additionally, antibodies vary by:

• Host species (rabbit, chicken, mouse)

• Clonality (polyclonal vs. monoclonal)

• Performance across applications (WB, IHC, IF)

• Cross-reactivity profiles (human, mouse, rat)

When selecting a PANX1 antibody, consider the specific requirements of your experiment and available validation data.

How do I troubleshoot inconsistent results when using PANX1 antibodies in Western blots?

When encountering inconsistent results with PANX1 antibodies in Western blots, implement these troubleshooting strategies:

• Sample preparation optimization:

  • Ensure complete protein denaturation

  • Use fresh reducing agents

  • Try different lysis buffers optimized for membrane proteins

  • Include protease inhibitors to prevent degradation

• Antibody-specific adjustments:

  • Test a range of dilutions (1:1000-1:6000 is recommended for many commercial antibodies)

  • Optimize blocking conditions (milk vs. BSA)

  • Try longer primary antibody incubation times (overnight at 4°C)

  • Adjust membrane transfer conditions for optimal protein transfer

• Address glycosylation heterogeneity:

  • Treat samples with PNGase F to remove N-linked glycans

  • Run gradient gels to better separate different glycosylated forms

  • Include positive controls with known glycosylation patterns

• Controls and validation:

  • Include tissues known to express PANX1 (brain, spleen)

  • Use knockout tissue when available

  • Consider using tagged PANX1 overexpression as a reference

  • Run multiple antibodies targeting different epitopes

Remember that PANX1 often appears as multiple bands due to various glycosylation states and potential processing forms. As noted in research: "Every antibody tested, even CT-395 (which Bargiotas et al. stated was the one successful antibody to show knockout of Panx1), showed additional bands that are not eliminated or reduced in a KO tissue" .

How does PANX1 localization vary across different brain regions?

PANX1 shows distinct localization patterns across brain regions, with both regional and cellular differences:

Subcellular localization varies by antibody: those against the intracellular loop and C-terminus preferentially label cell bodies, while N-terminal antibodies highlight neuronal processes. This may reflect different accessible PANX1 populations or specificity variations .

What is the current understanding of PANX1 channel properties and ionic selectivity?

The fundamental properties of PANX1 channels have been subjects of scientific debate, with recent studies challenging earlier views:

Channel Conductance:

• Early studies reported a high unitary conductance (~500 pS) with multiple sub-conductance states in Xenopus oocytes

• Recent work identified a much smaller conductance (~70-75 pS) in mammalian cell lines

• These discrepancies remain unexplained but may relate to expression systems or activation states

Ionic Selectivity:

• Initially considered a non-selective channel allowing passage of both cations and anions

• Recent studies suggest greater anion selectivity based on reversal potential measurements with ion substitution

• Evidence for both perspectives exists:

  • Anion permeability: Reversal potential shifts with chloride replacement but not Na+/NMDG+ substitution

  • Cation permeability: Both cationic and anionic dyes can flux across the membrane during activation

Permeability to Large Molecules:

• Permits passage of fluorescent dyes (To-Pro, Yo-Pro, fluorescein, Lucifer Yellow)

• Allows ATP release, though direct measurement of ATP permeation through single channels is limited

• Permeability may change during sustained stimulation, potentially through a pore dilation mechanism similar to TRP channels

These unresolved questions highlight the need for further research on basic PANX1 channel properties.

How do PANX1 knockout models affect antibody validation experiments?

PANX1 knockout models are crucial for antibody validation but present several complexities:

• Knockout strategy variations:

  • Complete gene deletion vs. exon-specific knockouts

  • "Knockout-first" strategy can result in hypomorphs rather than true knockouts

  • Potential for truncated proteins or alternative splicing products

• Observed Western blot patterns:

  • Reduction/elimination of bands in 36-55 kDa region (expected PANX1 size)

  • Some antibodies show reduction in ~20 kDa bands

  • Residual weak bands may indicate incomplete knockdown

  • Different antibodies show variable reduction patterns in identical knockout tissues

• Tissue-specific and model-dependent variations:

  • Same antibody may show different patterns in different tissues from the same animal

  • Variations observed between different knockout models (e.g., Genentech KO vs. KOMP KO)

  • "Every antibody tested... showed additional bands that are not eliminated or reduced in a KO tissue"

These complexities underscore the importance of using multiple validation approaches and carefully interpreting results with knockout models. Quantitative PCR analysis demonstrated that some PANX1 knockout animals are hypomorphs rather than true knockouts .

What are potential therapeutic applications targeting PANX1 channels?

PANX1 channels represent promising therapeutic targets, particularly for inflammatory conditions:

• Anti-inflammatory applications:

  • PANX1 inhibition reduces ATP release, which mediates inflammatory signaling

  • Beneficial effects have been shown in inflammasome activation and various inflammatory diseases

  • PANX1 channel blockade improves outcomes in models of seizure, where deletion or blocking "reduced the amount of ATP that is released and improves the behavioral manifestation of seizures"

• Current inhibitory approaches:

  • Mimetic peptide inhibitors: 10Panx1 peptide targets the first extracellular loop (EL1)

  • Non-selective channel blockers: carbenoxolone, probenecid

  • Monoclonal antibodies and mini-antibody-based inhibition

• Clinical relevance:

  • Expression alterations observed in asthmatic patients

  • Potential applications in cardiovascular disease (α-adrenergic vasoconstriction)

  • Promising results in models of HIV replication and breast cancer metastasis

• Challenges and limitations:

  • Potential off-target effects (10Panx1 also inhibits Cx46 current)

  • Low plasma stability of peptide inhibitors

  • Need for development of more selective inhibitors with improved pharmacokinetics

Research suggests that "mini-antibody-based inhibition of Panx1 channels" may offer new therapeutic approaches for diseases involving inflammation or cell death .

How can I distinguish between different glycosylation states of PANX1?

Distinguishing different glycosylation states of PANX1 requires specific technical approaches:

• Deglycosylation enzymes:

  • PNGase F removes all N-linked glycans

  • Endoglycosidase H (Endo H) removes only high-mannose glycans

  • Enzyme treatment followed by Western blot shows mobility shifts

• Optimized electrophoresis conditions:

  • Gradient gels (4-12% or 4-20%) provide better separation

  • Extended run times improve resolution of closely migrating glycoforms

  • Silver staining may detect less abundant glycoforms

• Glycosylation-specific controls:

  • Site-directed mutagenesis of N-glycosylation sites (N254, N104, N91 in human PANX1)

  • Cells treated with glycosylation inhibitors (tunicamycin, swainsonine)

  • Comparison with recombinant protein expressed in glycosylation-deficient cells

• Functional correlation approaches:

  • Surface biotinylation to identify which glycoforms reach the plasma membrane

  • Subcellular fractionation to determine localization of different glycoforms

  • Correlation with channel function using electrophysiology or ATP release assays

PANX1 typically exists in three main glycosylation states: Gly0 (non-glycosylated, ~43 kDa), Gly1 (core glycosylated), and Gly2 (complex glycosylated, ~50 kDa). Understanding these states is important as they affect trafficking, channel function, and antibody recognition .

What protocols are recommended for PANX1 immunohistochemistry in brain tissue?

For optimal PANX1 immunohistochemistry in brain tissue, follow these protocol recommendations:

Tissue Preparation:

• Perfusion fixation with 4% paraformaldehyde

• Post-fixation: 24-48 hours at 4°C

• Cryoprotection in sucrose gradient before freezing

• Section thickness: 30-50 μm for brain regions

Antigen Retrieval:

• Heat-mediated retrieval with citrate buffer (pH 6.0)

• 10-30 minutes at sub-boiling temperature

Blocking and Permeabilization:

• 10% normal serum (matching secondary antibody host)

• 0.3% Triton X-100 for membrane permeabilization

• 1-3% BSA to reduce background

Antibody Incubation:

• Primary antibody dilutions typically 1:250 to 1:1000

• Incubate at 4°C for 24-48 hours with gentle agitation

• Extensive washing between steps (3-5 washes of 15 minutes each)

Detection and Imaging:

• Fluorescent secondary antibodies for co-localization studies

• Include DAPI or other nuclear counterstain

• Use confocal microscopy for subcellular localization

• Consider wide field mosaic imaging for large-scale regional analysis while maintaining resolution

Essential Controls:

• Peptide competition controls to confirm specificity

• Omit primary antibody as negative control

• Include tissues from knockout animals when available

Different brain regions and specific antibodies may require optimization of these parameters.

What methodological approaches best correlate PANX1 channel activity with protein expression?

Correlating PANX1 channel activity with protein expression requires integrated methodological approaches:

Additionally, researchers should consider:

• Post-translational modification analysis:

  • Site-directed mutagenesis of key residues

  • Pharmacological manipulation of modifications

  • Western blot analysis to correlate modifications with function

• Advanced imaging approaches:

  • FRET-based reporters for conformational changes

  • Super-resolution microscopy for channel clustering

  • Single-molecule tracking for dynamics

• Mathematical modeling:

  • Correlative models linking expression to predicted activity

  • Kinetic models incorporating post-translational regulation

These integrated approaches provide comprehensive understanding of the relationship between PANX1 expression and function, addressing current questions in the field about channel properties and activation mechanisms .

What are the unresolved questions about PANX1 channel structure and function?

Despite significant advances in PANX1 research, several fundamental questions remain unresolved:

• Channel permeability and selectivity:

  • Is PANX1 strictly anion-selective or does it allow cation permeation under certain conditions?

  • Does channel selectivity change during different activation states?

  • What is the relationship between ionic current, ATP release, and dye uptake?

• Unitary conductance discrepancies:

  • Why do different studies report such different single-channel conductances (~70 pS vs. ~500 pS)?

  • Are conductance differences related to expression systems, activation mechanisms, or experimental conditions?

• Activation mechanisms:

  • How many C-termini must be cleaved to relieve inhibition of multimeric channels?

  • Are the requirements the same for ionic current, ATP release, and dye uptake?

  • Do different activation stimuli (receptor-mediated, mechanical stress, apoptosis) affect channel properties differently?

• Structural dynamics:

  • Does PANX1 undergo pore dilation during sustained activation like some TRP channels?

  • How do individual subunits contribute to channel gating and permeation?

  • What is the precise mechanism of PANX1 inhibition by various blockers?

As noted in recent research: "These new observations raise a number of questions regarding long-held views on the basic properties of Panx1 channels" , highlighting the need for further investigation using advanced techniques like cryo-electron microscopy and high-resolution electrophysiology.

How does PANX1 research intersect with clinical and disease-related investigations?

PANX1 research increasingly connects with various clinical and disease-related investigations:

Future clinical research directions include:

• Development of PANX1-selective channel blockers based on recent structural insights

• Exploration of mini-antibody-based PANX1 inhibition for therapeutic applications

• Investigation of PANX1 as a biomarker for diseases including asthma, where ROC curve analysis showed Panx1 expression could identify asthmatic patients

• Characterization of PANX1 polymorphisms and their association with disease susceptibility

As our understanding of PANX1 biology deepens, the potential for therapeutic targeting of these channels in various pathological conditions continues to expand.