PARD6G Antibody

What is PARD6G and what is its biological significance?

PARD6G (Par-6 family cell polarity regulator gamma) is an adapter protein critically involved in asymmetrical cell division and cell polarization processes. The canonical human PARD6G protein consists of 376 amino acid residues with a molecular mass of approximately 40.9 kDa . It is primarily localized to the cell membrane and cytoplasm, with up to two different isoforms reported . PARD6G belongs to the PAR6 protein family and functions as a component of the Par6/aPKC/Cdc42 polarity protein complex, which regulates polarization processes during epithelial morphogenesis, astrocyte migration, and axon specification .

This protein exhibits widespread expression throughout human tissues, with notably higher expression in both fetal and adult kidney tissues . The PARD6G gene has orthologs in multiple species including mouse, rat, bovine, frog, chimpanzee, and chicken, indicating evolutionary conservation and biological importance . Understanding PARD6G function is particularly relevant to research on epithelial cell polarity, development, and certain pathological conditions, including cancer progression.

What applications are PARD6G antibodies suitable for in research?

PARD6G antibodies are versatile research tools applicable across multiple experimental techniques. Based on manufacturer specifications, these antibodies are validated for Western Blotting (WB), which is the most widely used application for detecting and quantifying PARD6G protein expression levels . They are also suitable for various immunostaining techniques, including Immunohistochemistry (IHC) with both frozen and paraffin-embedded sections, Immunocytochemistry (ICC), and Immunofluorescence (IF) .

Additionally, many PARD6G antibodies are validated for Enzyme-Linked Immunosorbent Assay (ELISA) applications . The specific dilution requirements vary by application: for Western Blot, recommended concentrations are around 0.4 μg/ml; for Immunohistochemistry, Immunocytochemistry, and Immunofluorescence, 1-4 μg/ml is typically recommended; and for paraffin-embedded IHC, dilutions ranging from 1:50 to 1:200 are suggested . These applications allow researchers to investigate PARD6G expression, localization, and interactions in various experimental contexts.

What types of PARD6G antibodies are commercially available?

Multiple formats of PARD6G antibodies are commercially available to suit different experimental needs. The most common format is unconjugated (primary) antibodies, which require a secondary detection system . These are typically rabbit polyclonal antibodies raised against specific regions of the human PARD6G protein .

For specialized applications, conjugated versions are available including:

• FITC (fluorescein isothiocyanate) conjugated antibodies for direct fluorescence detection

• HRP (horseradish peroxidase) conjugated antibodies for enhanced chemiluminescence detection

• Biotin-conjugated antibodies for streptavidin-based detection systems

• APC (allophycocyanin) conjugated antibodies for flow cytometry applications

These antibodies target different epitopes of the PARD6G protein, with some specifically recognizing N-terminal regions (amino acids 18-47) and others targeting C-terminal regions (amino acids 249-326) . This diversity allows researchers to select antibodies appropriate for their specific experimental design and biological questions.

How do mutations in the PARD6G binding domains affect cell polarity and proliferation?

Research investigating the mechanisms of Par6-induced cell proliferation has revealed critical insights through mutational analysis of binding domains. Par6 contains several functional domains that mediate its interactions with other members of the polarity complex, including a PB1 (Phox/Bem1p) domain that binds to aPKC, a semi-CRIB domain that interacts with Cdc42, and a PDZ domain involved in binding to Lgl .

Experimental evidence shows that mutations disrupting these interactions significantly impact Par6 function. Specifically, a Lysine to Alanine mutation (K19A) in the PB1 domain abolishes binding to aPKC, deletion of Proline 136 (ΔPro136) in the semi-CRIB domain disrupts Cdc42 binding, and substitution of Methionine with Tryptophan (M235W) in the PDZ domain impairs Lgl binding . These mutations have been shown to affect Par6's ability to induce cell proliferation, suggesting that interactions with aPKC and Cdc42 are particularly crucial for its proliferative effects.

Understanding these domain-specific interactions is essential for researchers investigating how PARD6G contributes to cell polarity establishment and maintenance, as well as its role in pathological conditions characterized by dysregulated proliferation, such as cancer.

What is the role of PARD6G in cancer research and how can antibodies help investigate it?

PARD6G and related family members have emerged as significant factors in cancer research, particularly in breast cancer. While studies have shown that Pard6b is amplified in breast cancer, the role of Par6 overexpression during transformation of breast epithelial cells is an active area of investigation . Research utilizing antibodies against Par6 has revealed that overexpression of Par6 isoforms (Par6α and Par6β) in mammary epithelial cells affects three-dimensional morphogenesis and proliferation patterns.

Experimental evidence shows that Par6 overexpression induces the development of hyperplastic acini by inhibiting proliferation arrest during 3D morphogenesis without disrupting polarity . This was demonstrated through studies of proliferation markers like Ki-67, which showed higher proliferation rates in Par6-expressing cells compared to controls . Importantly, this effect was not associated with inhibition of cell death pathways.

PARD6G antibodies are instrumental in such research for:

• Detecting changes in PARD6G expression levels in normal versus cancer tissues

• Visualizing alterations in subcellular localization during cancer progression

• Assessing protein-protein interactions between PARD6G and other polarity complex members

• Evaluating the effectiveness of potential therapeutic interventions targeting PARD6G-mediated pathways

These applications make PARD6G antibodies valuable tools for researchers investigating the fundamental mechanisms of cancer development and potential therapeutic targets.

How can researchers validate the specificity of PARD6G antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For PARD6G antibodies, several validation approaches should be considered:

• Peptide competition assays: Preincubating the antibody with the immunizing peptide (such as the synthetic peptide corresponding to amino acids 18-47 or other specified regions) should block specific binding in subsequent applications . This serves as a critical control to confirm binding specificity.

• Knockout/knockdown controls: Using PARD6G knockout cell lines or cells treated with PARD6G-specific siRNA/shRNA as negative controls can convincingly demonstrate antibody specificity. The absence or reduction of signal in these samples compared to wild-type cells provides strong evidence of specificity.

• Multiple antibody validation: Using multiple antibodies targeting different epitopes of PARD6G should yield consistent results for truly specific antibodies. Some commercially available antibodies have been specifically verified on protein arrays containing target protein plus 383 other non-specific proteins to ensure specificity .

• Recombinant protein controls: Testing the antibody against purified recombinant PARD6G protein can provide information about sensitivity and specificity. Several available antibodies are generated against recombinant protein corresponding to specific amino acid sequences of PARD6G .

• Cross-reactivity testing: Examining potential cross-reactivity with other PAR family members (PARD6A, PARD6B) is essential, particularly when studying tissues or cells that express multiple family members.

Proper validation ensures that experimental observations genuinely reflect PARD6G biology rather than artifacts or cross-reactivity.

What are the optimal protocols for using PARD6G antibodies in Western blot analysis?

For optimal Western blot analysis using PARD6G antibodies, researchers should follow these methodological guidelines:

Sample Preparation:

• Extract total protein from cells or tissues using standard lysis buffers containing protease inhibitors

• Determine protein concentration using Bradford or BCA assay

• Prepare samples containing 20-50 μg of total protein with reducing sample buffer

• Heat samples at 95°C for 5 minutes to denature proteins

Electrophoresis and Transfer:

• Resolve proteins on 10-12% SDS-PAGE gels (appropriate for the 40.9 kDa PARD6G protein)

• Transfer proteins to PVDF or nitrocellulose membranes

Antibody Incubation:

• Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary PARD6G antibody at the recommended concentration of 0.4 μg/ml

• Dilute antibody in blocking buffer and incubate overnight at 4°C

• Wash membrane 3-5 times with TBST

• Incubate with appropriate HRP-conjugated secondary antibody (if using unconjugated primary)

• Develop using enhanced chemiluminescence detection

Controls:

• Include positive control samples known to express PARD6G

• Include molecular weight markers to confirm band size (expected around 40.9 kDa)

• Consider running a peptide-competition control to verify specificity

This protocol should allow for specific detection of PARD6G protein while minimizing background and non-specific binding. Optimization may be required for specific sample types or experimental conditions.

How should researchers approach immunohistochemistry with PARD6G antibodies?

Immunohistochemistry (IHC) with PARD6G antibodies requires careful attention to methodology for optimal results:

For Paraffin-Embedded Sections:

• Tissue Processing:

  • Fix tissues in 10% neutral-buffered formalin for 24-48 hours

  • Process, embed in paraffin, and section at 4-6 μm thickness

• Antigen Retrieval:

  • Deparaffinize and rehydrate sections

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Maintain temperature at 95-98°C for 15-20 minutes

• Antibody Incubation:

  • Block endogenous peroxidase with 3% H₂O₂

  • Apply protein block to reduce background

  • Dilute PARD6G antibody at 1:50 to 1:200 as recommended

  • Incubate at 4°C overnight or at room temperature for 1-2 hours

  • Use appropriate detection system (e.g., polymer-HRP and DAB chromogen)

  • Counterstain with hematoxylin

For Frozen Sections:

• Tissue Processing:

  • Snap-freeze tissue in OCT compound

  • Section at 5-10 μm thickness

  • Fix briefly in cold acetone or 4% paraformaldehyde

• Antibody Incubation:

  • Block with serum-containing buffer

  • Apply PARD6G antibody at 1-4 μg/ml concentration

  • Incubate overnight at 4°C

  • Apply appropriate detection system

Controls and Validation:

• Include positive control tissues with known PARD6G expression

• Include negative controls (omitting primary antibody)

• Consider kidney sections as positive controls, given the reported higher expression in this tissue

For multiplexing with other antibodies, sequential staining protocols or spectral imaging systems may be necessary. Optimization of dilution, incubation time, and antigen retrieval conditions is recommended for each specific antibody and tissue type.

What considerations are important for immunofluorescence experiments with PARD6G antibodies?

For successful immunofluorescence (IF) experiments using PARD6G antibodies, researchers should consider the following methodological approaches:

Sample Preparation:

• Cell Culture:

  • Grow cells on glass coverslips or chamber slides

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Tissue Sections:

  • For frozen sections, fix briefly with cold acetone or methanol

  • For paraffin sections, perform antigen retrieval after deparaffinization

Antibody Incubation Protocol:

• Block non-specific binding with 5% normal serum from the same species as the secondary antibody

• Dilute PARD6G antibody to 1-4 μg/ml in blocking buffer

• Incubate overnight at 4°C in a humidified chamber

• Wash thoroughly with PBS (3-5 times, 5 minutes each)

• Apply appropriate fluorochrome-conjugated secondary antibody (if using unconjugated primary)

• For direct detection, use FITC-conjugated PARD6G antibodies at manufacturer-recommended dilutions

• Include DAPI or other nuclear counterstain

• Mount with anti-fade mounting medium

Co-localization Studies:

• PARD6G is known to interact with aPKC and Cdc42 as part of the polarity complex

• Use antibodies against these proteins for co-localization studies

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

• Use appropriate controls to confirm specificity of co-localization signals

Image Acquisition and Analysis:

• Use confocal microscopy for precise subcellular localization

• Capture z-stacks to analyze distribution in all dimensions

• Apply consistent imaging parameters across experimental and control samples

• For quantitative analysis, use appropriate software to measure signal intensity and co-localization coefficients

Given PARD6G's role in cell polarity, pay particular attention to its distribution at cell-cell junctions, cytoplasmic regions, and membrane domains when interpreting results.

How can researchers design experiments to investigate PARD6G interactions with other polarity complex proteins?

Designing experiments to study PARD6G interactions with other polarity complex members requires careful planning and multiple complementary approaches:

Co-immunoprecipitation (Co-IP) Design:

• Antibody Selection:

  • Choose PARD6G antibodies specifically validated for immunoprecipitation

  • Ensure the antibody epitope does not overlap with binding domains for interaction partners

• Protocol Design:

  • Prepare cell lysates under non-denaturing conditions to preserve protein-protein interactions

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Incubate cleared lysates with PARD6G antibody (or control IgG)

  • Isolate immune complexes and analyze by Western blot using antibodies against suspected interaction partners (aPKC, Cdc42, Par3, Lgl)

• Controls:

  • Include IgG control immunoprecipitations

  • Consider using cells expressing PARD6G mutants defective in specific interactions as negative controls

Proximity Ligation Assay (PLA) Approach:

• Use antibodies against PARD6G and potential binding partners

• PLA signal will only be generated if proteins are within 40 nm of each other

• This technique can visualize interactions in situ without overexpression

Domain Mapping Experiments:

• Generate PARD6G constructs with specific domain mutations:

  • K19A mutation in PB1 domain (disrupts aPKC binding)

  • ΔPro136 in CRIB domain (disrupts Cdc42 binding)

  • M235W in PDZ domain (disrupts Lgl binding)

• Perform functional assays to determine how these mutations affect:

  • Localization of PARD6G

  • Cell polarity establishment

  • Proliferation phenotypes

Advanced Imaging Approaches:

• FRET Analysis:

  • Generate fluorescently tagged PARD6G and interaction partner constructs

  • Measure energy transfer as indication of direct protein interaction

  • Compare wild-type with domain mutants

• Live-Cell Imaging:

  • Monitor dynamics of PARD6G-GFP and interaction partners during polarity establishment

  • Combine with domain mutants to assess functional significance

These approaches collectively provide strong evidence for physical and functional interactions between PARD6G and polarity complex components.

What are the common issues encountered when using PARD6G antibodies and how can they be resolved?

Researchers working with PARD6G antibodies may encounter several technical challenges that can be addressed through systematic troubleshooting:

Issue 1: Weak or Absent Signal in Western Blot

• Potential Causes:

  • Insufficient protein expression

  • Degradation during sample preparation

  • Inefficient transfer of protein

  • Suboptimal antibody concentration

• Solutions:

  • Increase protein loading (50-100 μg total protein)

  • Add fresh protease inhibitors to lysis buffer

  • Verify transfer efficiency with reversible stain

  • Optimize antibody concentration beyond the recommended 0.4 μg/ml

  • Try alternative detection systems with higher sensitivity

  • Extend exposure time during imaging

Issue 2: Multiple Bands or Non-specific Binding

• Potential Causes:

  • Cross-reactivity with related proteins

  • Sample degradation

  • Post-translational modifications

  • Alternative splice variants (up to 2 isoforms reported)

• Solutions:

  • Increase blocking time and concentration

  • Perform peptide competition controls

  • Use freshly prepared samples

  • Try antibodies targeting different epitopes

  • Increase washing stringency (higher salt concentration, longer wash times)

Issue 3: Background in Immunohistochemistry or Immunofluorescence

• Potential Causes:

  • Inadequate blocking

  • Excessive antibody concentration

  • Non-specific secondary antibody binding

  • Autofluorescence (in IF)

• Solutions:

  • Extend blocking time to 2 hours

  • Titrate antibody concentration (starting with 1:200 dilution for IHC-p)

  • Include 0.1-0.3% Triton X-100 in antibody diluent

  • For IHC: treat sections with hydrogen peroxide before antibody incubation

  • For IF: include Sudan Black B treatment to reduce autofluorescence

  • Test alternative blocking reagents (BSA, normal serum, commercial blockers)

Issue 4: Inconsistent Results Across Experiments

• Potential Causes:

  • Antibody degradation

  • Variable fixation conditions

  • Batch-to-batch antibody variation

• Solutions:

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Store at 4°C short term or -20°C long term with minimal freeze-thaw cycles

  • Standardize fixation protocols across experiments

  • Include internal positive controls in each experiment

  • Document lot numbers and validate new antibody batches against previous results

By applying these systematic troubleshooting approaches, researchers can optimize their experiments with PARD6G antibodies and obtain reliable, reproducible results.

How can researchers design experiments to investigate PARD6G's role in cellular proliferation?

Based on findings that Par6 overexpression affects cell proliferation during 3D morphogenesis , researchers can design comprehensive experiments to investigate PARD6G's role in proliferation:

Experimental Design Approach:

• Cell Model Selection:

  • Use non-transformed epithelial cell lines (e.g., MCF-10A breast epithelial cells)

  • Compare with cancer cell lines with known polarity defects

  • Consider primary cells from relevant tissues with high PARD6G expression (e.g., kidney)

• Manipulation of PARD6G Expression:

  • Overexpression Studies:

    • Generate stable cell lines expressing wild-type PARD6G

    • Include domain mutants (K19A, ΔPro136, M235W) to identify critical interaction domains

    • Use inducible expression systems for temporal control

  • Knockdown/Knockout Approaches:

    • Design siRNA/shRNA targeting PARD6G

    • Implement CRISPR-Cas9 genome editing for complete knockout

    • Consider conditional systems for developmental studies

• 3D Culture Systems:

  • Embed cells in Matrigel to form acini-like structures

  • Monitor morphogenesis over 20+ days

  • Compare acinar size, lumen formation, and polarization between control and PARD6G-manipulated cells

• Proliferation Assessment Methods:

  • Immunostaining for Proliferation Markers:

    • Monitor Ki-67 expression at multiple timepoints (days 8, 12, 16, 20)

    • Assess BrdU or EdU incorporation for S-phase analysis

    • Quantify phospho-histone H3 for mitotic index

  • Cell Counting:

    • Determine cell number per acinus at defined timepoints

    • Use automated image analysis for unbiased quantification

  • Live Imaging:

    • Express fluorescent cell cycle indicators

    • Track division patterns in real-time during morphogenesis

• Cell Death Analysis:

  • Assess whether changes in acinar size result from proliferation or reduced apoptosis

  • Use TUNEL staining or cleaved caspase-3 immunostaining

  • Compare wild-type and mutant PARD6G effects on cell death pathways

• Signaling Pathway Investigation:

  • Examine activation state of proliferation-related pathways

  • Assess MAPK, PI3K/Akt, and Wnt signaling

  • Use phospho-specific antibodies to monitor pathway activation

• Rescue Experiments:

  • In PARD6G-depleted cells, reintroduce wild-type or mutant constructs

  • Assess which domains are essential for proliferation phenotypes

  • Determine whether other PAR6 family members can compensate

This comprehensive experimental design allows researchers to systematically investigate PARD6G's role in cellular proliferation while providing mechanistic insights into domain requirements and downstream effectors.

What are emerging techniques for studying PARD6G localization and dynamics in live cells?

As research on PARD6G continues to evolve, several cutting-edge techniques offer new opportunities for studying its localization and dynamics in live cells:

Advanced Fluorescence Microscopy Approaches:

• FRAP (Fluorescence Recovery After Photobleaching):

  • Tag PARD6G with fluorescent proteins

  • Photobleach specific cellular regions

  • Measure recovery rate to determine mobility and binding dynamics

  • Compare dynamics at different subcellular locations (cell-cell junctions vs. cytoplasm)

• Super-Resolution Microscopy:

  • Apply STORM, PALM or STED microscopy for nanoscale resolution

  • Visualize PARD6G organization within polarity complexes

  • Resolve distribution at membrane microdomains

  • Combine with proximity labeling for interaction studies

• Lattice Light-Sheet Microscopy:

  • Enable long-term imaging with minimal phototoxicity

  • Capture rapid 3D dynamics during polarity establishment

  • Follow PARD6G localization during cell division in developing tissues

Emerging Protein Tagging Strategies:

• Split Fluorescent Protein Complementation:

  • Tag PARD6G and interaction partners with complementary fragments

  • Fluorescence only occurs upon protein-protein interaction

  • Map spatial distribution of specific complexes

• HaloTag or SNAP-tag Technologies:

  • Create PARD6G fusion proteins compatible with multiple labeling strategies

  • Allow pulse-chase experiments to track protein turnover

  • Compatible with cell-permeable fluorescent ligands for live-cell imaging

• Optogenetic Control of PARD6G:

  • Engineer light-responsive PARD6G variants

  • Control localization or activity with specific wavelengths of light

  • Study temporal requirements for PARD6G function

These advanced techniques can provide unprecedented insights into PARD6G dynamics, helping researchers understand how this protein contributes to cell polarity establishment and maintenance across different cellular contexts and disease states.

How can multi-omics approaches enhance our understanding of PARD6G function?

Integrating multiple -omics approaches offers powerful opportunities to comprehensively understand PARD6G function:

Proteomics Applications:

• Interactome Analysis:

  • Perform immunoprecipitation-mass spectrometry (IP-MS) with PARD6G antibodies

  • Identify novel interaction partners beyond known polarity proteins

  • Compare interactomes under different cellular conditions (polarized vs. non-polarized)

• Post-Translational Modification (PTM) Mapping:

  • Use phospho-proteomics to identify regulatory phosphorylation sites

  • Determine how PTMs affect PARD6G interactions and function

  • Investigate ubiquitination, SUMOylation and other modifications

• Proximity Labeling Proteomics:

  • Generate PARD6G-BioID or APEX2 fusion proteins

  • Map protein neighborhoods in different subcellular compartments

  • Identify transient or context-specific interactions

Transcriptomics Approaches:

• RNA-Seq After PARD6G Manipulation:

  • Compare gene expression profiles in PARD6G-overexpressing vs. control cells

  • Identify downstream transcriptional programs affected by PARD6G

  • Correlate with proliferation phenotypes observed in 3D culture

• Single-Cell Transcriptomics:

  • Analyze cell populations with varying PARD6G expression levels

  • Identify cell state transitions associated with PARD6G function

  • Combine with spatial transcriptomics for tissue context

Functional Genomics Integration:

• CRISPR Screens:

  • Conduct synthetic lethality screens in PARD6G-manipulated backgrounds

  • Identify genetic dependencies and compensatory pathways

  • Map genetic interaction networks

• Chromatin Organization:

  • Investigate whether PARD6G affects nuclear organization

  • Study links between cell polarity and gene expression regulation

  • Apply Hi-C or similar techniques to map 3D genome organization

Systems Biology Integration:

• Computational Modeling:

  • Develop mathematical models of polarity complex assembly

  • Simulate effects of PARD6G perturbation on cell polarity establishment

  • Generate testable predictions about system behavior

• Multi-Modal Data Integration:

  • Combine proteomics, transcriptomics, and imaging data

  • Identify emergent properties not apparent in single-omics approaches

  • Apply machine learning to recognize patterns across datasets

These multi-omics strategies can provide a systems-level understanding of PARD6G function, revealing how this polarity regulator integrates into broader cellular networks and identifying potential intervention points for diseases involving polarity dysregulation.