PANX2 Antibody

What is PANX2 and why is it significant in biological research?

PANX2 is a member of the pannexin family of putative single membrane channels discovered through homology to invertebrate gap junction-forming proteins called innexins. It combines with Pannexin 1 to form cell type-specific gap junctions with distinct properties. PANX2 is significant because it is differentially expressed by multipotential progenitor cells and mature neurons in the postnatal hippocampus, suggesting a role in neural development and function . Additionally, PANX2 has been associated with conditions such as Oculodentodigital Dysplasia and Chronic Closed-Angle Glaucoma . Unlike earlier assumptions of CNS-specific expression, recent research reveals PANX2 protein is present at substantial levels in multiple tissues throughout the body, indicating broader physiological significance than initially thought .

Which validated PANX2 antibodies are available for research applications?

Several well-characterized PANX2 antibodies have been validated for research applications:

• Monoclonal antibodies:

  • Clone N121A/1: A mouse monoclonal antibody that recognizes the last 15 amino acids of the PANX2 C-terminal tail

  • Clone N121A/31: Another highly specific mouse monoclonal antibody for PANX2

• Polyclonal antibodies:

  • Anti-PANX2 polyclonal antibody (Aviva Systems Biology)

  • Anti-PANX2 C-term polyclonal antibody targeting the C-terminal region

The monoclonal antibodies were generated using an immunogen comprising the entire rat PANX2 protein sequence (accession number P60571) minus the first 10 amino acids . Both clones have been rigorously validated for specificity against PANX1 and PANX2 in Western blot, immunofluorescence, and immunohistochemistry applications .

How can researchers verify the specificity of PANX2 antibodies?

Verifying PANX2 antibody specificity is critical for generating reliable research data. Multiple approaches should be employed:

• Western blot validation:

  • Both N121A/1 and N121A/31 clones identify a single band of approximately 70 kDa in cells expressing PANX2

  • Neither clone cross-reacts with PANX1, which is often co-expressed with PANX2 in the CNS

  • When PANX2 is tagged with GFP, the detected band shifts by ~27 kDa, confirming specificity

• Immunofluorescence validation:

  • Labeling should overlap with GFP fluorescence signal in PANX2-GFP expressing cells but not with PANX1-GFP

  • Use immunoglobulin from non-immunized mouse as a negative control to rule out non-specific binding

• Endogenous expression confirmation:

  • Antibodies should recognize a ~70 kDa band in tissues known to express PANX2

  • Compare staining patterns with published literature on PANX2 distribution

• Multiple detection methods:

  • Concordance between RT-PCR, Western blot, and immunostaining increases confidence in antibody specificity

What are the optimal protocols for PANX2 immunodetection?

For robust PANX2 detection across different applications, these protocols are recommended:

• Western Blotting:

  • Antibody concentrations: Use anti-PANX2 monoclonal antibodies (N121A/1, N121A/31) at 5 μg/mL

  • Sample preparation: Employ RIPA or similar lysis buffer containing protease inhibitors

  • Detection: Look for primary band at ~70 kDa; be aware of potential lower molecular weight bands (~50 kDa)

• Immunofluorescence/Immunohistochemistry:

  • Antibody concentrations: Use monoclonal antibodies at 20 μg/mL

  • Tissue preparation: For frozen sections, use 10-μm cryosections

  • Antibody diluent: Dilute in 10 mM PBS with 0.3% Triton X-100 and 3% bovine serum albumin

  • Controls: Include isotype controls at matching concentrations

• Technical considerations:

  • Storage: Aliquot and store at ≤ -20°C long-term; 2-8°C short-term

  • Epitope awareness: Clone N121A/1 recognizes the last 15 amino acids of the C-terminal tail, so C-terminal tags may interfere with binding

  • Cross-reactivity: Confirmed that the described monoclonal antibodies do not cross-react with PANX1 or PANX3

How does PANX2 expression vary across different tissue types?

PANX2 expression shows notable tissue-specific patterns that challenge earlier assumptions:

• Central Nervous System:

  • Expressed by both multipotential progenitor cells and mature neurons in postnatal hippocampus

  • Contrary to initial beliefs, some studies suggest PANX2 protein levels may actually be lower in the nervous system compared to other tissues

• Gastrointestinal Tract:

  • Strong PANX2 immunoreactivity in glandular and epithelial cells

  • In the stomach, parietal cells (which secrete gastric acid) and epithelial cells show robust PANX2 expression

  • In the small and large intestine, columnar epithelial cells are strongly PANX2-positive

• Additional Tissues:

  • Substantial PANX2 protein detected in eye, thyroid, prostate, kidney, and liver

  • Semi-quantitative analyses suggest PANX2 protein is present at substantial levels in every tissue studied

• Expression Level Discrepancies:

  • PANX2 mRNA and protein levels are not always correlated, suggesting post-transcriptional regulation

  • This mRNA-protein discrepancy highlights the importance of protein-level detection methods

What is the subcellular localization pattern of PANX2 compared to PANX1?

PANX2 and PANX1 display distinct subcellular localization patterns, suggesting different functional roles:

• PANX2 localization:

  • Primarily found in perinuclear and cytoplasmic regions

  • In epithelial cells of the gastrointestinal tract, PANX2 remains largely confined to the cytoplasmic area

  • Some studies suggest PANX2 localizes to endosomal vesicle membranes

• PANX1 localization:

  • Predominantly restricted to the plasma membrane

  • In epithelial cells, often found at cell-cell junctions

• Co-localization analysis:

  • In columnar epithelial cells of the colon, PANX1 and PANX2 do not co-localize

  • While PANX1 is found at the plasma membrane between epithelial cells, PANX2 is not discernible at the plasma membrane

• Developmental considerations:

  • Some evidence suggests PANX2 localization may change during development

  • Different localization patterns may exist between neural progenitors and mature neurons

This distinct subcellular distribution indicates that despite being in the same protein family, PANX1 and PANX2 likely serve different cellular functions.

What controls should be included when using PANX2 antibodies?

A comprehensive set of controls is essential for reliable PANX2 antibody experimentation:

• Primary controls:

  • Isotype control: Use immunoglobulin from non-immunized mouse at the same concentration as the PANX2 antibody

  • Positive expression control: Include samples known to express PANX2 (e.g., tissues from CNS or GI tract)

  • Negative expression control: Use PANX2 knockdown or knockout samples if available

• Secondary antibody controls:

  • Secondary-only control: Include samples treated only with secondary antibody (omitting primary)

  • Be aware that anti-mouse secondary antibodies may detect endogenous immunoglobulins in certain tissues, particularly lymphocytes

• Application-specific controls:

  • For Western blotting: Include molecular weight markers to confirm the expected size (~70 kDa)

  • For immunofluorescence: Include nuclear counterstains to aid interpretation of subcellular localization

  • For tagged constructs: Compare staining between anti-PANX2 and anti-tag antibodies, noting that C-terminal tags may interfere with C-terminus-targeting antibodies

How can researchers troubleshoot inconsistent PANX2 staining patterns?

When encountering inconsistent staining, consider these technical and biological factors:

• Technical variables:

  • Verify antibody concentration (20 μg/mL for immunofluorescence, 5 μg/mL for Western blotting)

  • Check antibody storage conditions (≤ -20°C long-term; 2-8°C short-term)

  • Standardize fixation and permeabilization protocols - overfixation can mask epitopes

  • Verify secondary antibody specificity and compatibility

• Biological considerations:

  • PANX2 expression varies significantly across tissues and may be lower in nervous system than initially expected

  • Account for cell type heterogeneity within tissues

  • Remember PANX2 localization is primarily perinuclear/cytoplasmic, not at the plasma membrane

• Systematic approach:

  • Validate with multiple methods (Western blot, immunofluorescence, RT-PCR)

  • Use multiple antibodies targeting different PANX2 epitopes

  • Adjust blocking conditions to reduce background and optimize detergent concentrations

  • Consider signal amplification methods for low expression samples

What is the expected molecular weight of PANX2 in Western blots?

The expected molecular weight of PANX2 in Western blots is approximately 70 kDa, though several important considerations apply:

• Standard detection:

  • Both endogenous PANX2 in tissue lysates and recombinant PANX2 in overexpression systems typically appear at ~70 kDa

  • This molecular weight has been consistently observed across different studies

• Tagged constructs:

  • When PANX2 is tagged with GFP at the C-terminal, molecular weight increases by ~27 kDa, resulting in a band at ~97 kDa

  • Other epitope tags will alter the apparent molecular weight proportionally to their size

• Additional bands:

  • Some studies report occasional detection of a ~50 kDa band in certain tissues

  • This may represent either PANX2 degradation products or detection of endogenous immunoglobulin heavy chains by anti-mouse secondary antibodies

  • Research indicates PANX2 can undergo caspase cleavage with caspase 3 or 7 treatment, yielding a ~36 kDa band

• Post-translational modifications:

  • PANX2 may undergo tissue-specific post-translational modifications affecting apparent molecular weight

  • These modifications could contribute to discrepancies between expected and observed molecular weights

How can researchers differentiate between specific and non-specific PANX2 antibody binding?

Distinguishing specific PANX2 signals from non-specific binding requires multiple validation approaches:

• Experimental controls:

  • Use isotype controls (immunoglobulin from non-immunized mouse) at matching concentrations

  • Include secondary antibody-only controls to assess non-specific binding

  • When possible, use PANX2 knockdown/knockout samples as biological negative controls

  • Consider peptide competition assays to confirm specificity

• Signal characteristics:

  • Verify molecular weight in Western blots (specific PANX2 signal at ~70 kDa)

  • Confirm expected subcellular localization (primarily perinuclear and cytoplasmic)

  • Check if staining intensity correlates with expected expression patterns across tissues

• Advanced validation:

  • Compare results using multiple antibodies targeting different PANX2 epitopes

  • In overexpression systems, compare anti-PANX2 staining with tagged PANX2 or anti-tag antibody staining

  • Cross-species validation can increase confidence in specificity if consistent patterns are observed