PARD6B Antibody

What is PARD6B and what applications are PARD6B antibodies suitable for?

PARD6B (par-6 partitioning defective 6 homolog beta) is a member of the PAR6 family and serves as an adapter protein involved in asymmetrical cell division and cell polarization processes. It contains a PSD95/Discs-large/ZO1 (PDZ) domain, an OPR domain, and a semi-Cdc42/Rac interactive binding (CRIB) domain . This cytoplasmic protein is critical in regulating epithelial tight junction formation and maintenance of apical-basal polarity .

PARD6B antibodies are validated for multiple applications:

Many commercial antibodies have been validated in multiple published studies for WB, IHC, and IF applications .

What is the expected molecular weight of PARD6B, and how does this compare with observed bands in Western blots?

While the calculated molecular weight of PARD6B is 41 kDa, the observed molecular weight in Western blots typically ranges from 50-53 kDa . This discrepancy between calculated and observed weights is common for many proteins and may reflect post-translational modifications, protein folding properties, or other structural characteristics. When conducting Western blot analysis, it's advisable to use positive controls such as HeLa cells, HEK-293 cells, human placenta tissue, or PC-3 cells, all of which have been validated to express detectable levels of PARD6B .

What species reactivity can researchers expect with commercially available PARD6B antibodies?

Most commercial PARD6B antibodies demonstrate reactivity with human, mouse, and rat samples . Some antibodies are also predicted to react with additional species based on sequence homology:

When using PARD6B antibodies for novel applications or untested species, validation with appropriate positive and negative controls is essential .

What are the optimal immunohistochemistry conditions for PARD6B detection in tissue samples?

For successful immunohistochemical detection of PARD6B in tissue samples, consider the following methodological recommendations:

• Antigen retrieval: Use TE buffer pH 9.0 for optimal results. As an alternative, citrate buffer pH 6.0 may be used if TE buffer is not available .

• Dilution range: The recommended dilution for IHC applications is 1:50-1:500, though this should be optimized for specific tissue types .

• Validated tissues: PARD6B antibodies have shown positive IHC staining in mouse kidney tissue and human pancreas tissue . Paraffin-embedded human gastric cancer and liver cancer tissues have also been successfully stained using a 1:100 to 1:325 dilution .

• Detection method: For chromogenic detection, protocols using HRP-conjugated secondary antibodies have been validated. For fluorescent detection, secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 546 at 1:1000 dilution have been successfully employed .

• Controls: Always include positive control tissues and negative controls (omitting primary antibody) to validate staining specificity.

How should researchers optimize immunofluorescence protocols for PARD6B detection?

For optimal immunofluorescence detection of PARD6B, follow these methodological guidelines:

• Cell fixation: Fix cells in 4% paraformaldehyde in PBS for 20-30 minutes at room temperature .

• Permeabilization: After washing with PBS containing 0.1% Tween-20 (PBSw), permeabilize cells with 0.5% Triton X-100 in PBS for 15 minutes .

• Blocking: Block with 5% bovine serum albumin in PBSw to minimize non-specific binding .

• Antibody incubation: Incubate with primary PARD6B antibody (1:10-1:100 dilution) overnight at 4°C, followed by appropriate secondary antibody (1:1000) for 1-2 hours .

• Validated cell lines: HepG2 cells and MCF-7 cells have been successfully used for IF/ICC detection of PARD6B .

• Co-staining recommendations: For co-localization studies, PARD6B can be co-stained with markers such as CDH1 (E-cadherin), CLDN4 (claudin-4), TJP1 (ZO-1), or PRKCZ (aPKCζ) to study tight junction formation and polarity .

What methodological approaches can resolve challenges in PARD6B immunoprecipitation experiments?

For successful immunoprecipitation of PARD6B:

• Lysate preparation: Use freshly prepared cell lysates from validated cell lines like HEK-293 cells, which have been confirmed to express PARD6B at levels suitable for IP .

• Antibody amount: Use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate for optimal precipitation efficiency .

• Pre-clearing: To reduce non-specific binding, pre-clear lysates with protein A/G beads before adding the PARD6B antibody.

• Controls: Include an IgG control (same species as the PARD6B antibody) to identify non-specific binding. Additionally, include a positive control IP using an antibody against a known PARD6B interacting partner (e.g., PRKCZ or CDC42) .

• Validation: Confirm successful IP by Western blot analysis using the same or a different PARD6B antibody that recognizes a separate epitope .

• Co-IP applications: For studying protein-protein interactions, gentler lysis buffers (containing 0.5% NP-40 or 1% Triton X-100) are recommended to preserve protein complexes.

How can PARD6B antibodies be effectively used to investigate its role in cancer progression?

PARD6B has been implicated in cancer progression, particularly in breast cancer where the PARD6B gene is located in a region (20q13.13) that is frequently amplified . Researchers can employ the following methodological approaches:

• Amplification detection: Use FISH (fluorescence in situ hybridization) to detect PARD6B amplification in cancer cell lines and tissue samples. The PARD6B amplicon has been identified in breast cancer cell lines with copy numbers ranging from 7 to 27 .

• Expression correlation: Correlate PARD6B protein expression with amplification status using validated antibodies in Western blot and IHC applications .

• Subcellular localization: Examine PARD6B localization in normal versus cancer tissues using immunohistochemistry. In poorly differentiated tumors, PARD6B staining appears reduced and cytoplasmic compared to normal epithelium .

• Functional studies: Combine PARD6B antibody detection with siRNA-mediated knockdown to study effects on tight junction formation, cell polarity, and proliferation. Previous studies have shown that PAR6B overexpression can induce EGF-independent cell proliferation .

• Co-localization with markers: Use PARD6B antibodies in conjunction with markers of epithelial-mesenchymal transition and cell polarity to track cancer progression in tissue samples .

What strategies can resolve contradictory results when studying PARD6B localization in different developmental contexts?

When studying PARD6B during developmental processes like embryogenesis, researchers may encounter contradictory results due to context-dependent functions. Consider these methodological approaches:

• Developmental timing: PARD6B function is highly dependent on developmental stage. In early embryonic development, it is essential for trophectoderm formation and blastocyst cavity development . Precise documentation of developmental stages is critical.

• Isoform specificity: Ensure antibodies specifically detect PARD6B without cross-reactivity to related isoforms (PARD6A and PARD6G). While PARD6B is the major gene expressed during preimplantation development, other isoforms may predominate in different contexts .

• Fixation protocol optimization: Different fixation methods can significantly affect epitope accessibility and apparent localization. Compare multiple fixation protocols (4% paraformaldehyde, methanol, or acetone) to ensure consistent results .

• Knockdown validation: Validate antibody specificity using knockdown approaches. In one study, PARD6B expression was knocked down by microinjecting RNA interference constructs into zygotes, providing a negative control for antibody specificity .

• Multiple detection methods: Combine immunofluorescence with other detection methods like in situ hybridization or transgenic reporter systems to corroborate localization patterns.

How can researchers effectively use PARD6B antibodies to study protein-protein interactions within the PAR complex?

The PAR complex consists of multiple interacting proteins, including PARD6B, PARD3, atypical PKC (aPKC), and CDC42/Rac. To study these interactions:

• Co-immunoprecipitation strategy: Use PARD6B antibodies to immunoprecipitate native protein complexes, followed by Western blot analysis for interacting partners. Studies have shown that PARD6B interacts with aPKC through its PB1 domain and with CDC42 through its CRIB domain .

• Domain-specific interactions: To analyze which domains are responsible for specific interactions, use antibodies against different PARD6B domains or epitopes. For instance:

  • The PB1 domain (containing K19) interacts with aPKC

  • The semi-CRIB domain (containing P136) interacts with CDC42

  • The PDZ domain (containing M235) interacts with Lgl

• Proximity ligation assay: Use PARD6B antibodies in conjunction with antibodies against potential interacting partners in proximity ligation assays to visualize protein-protein interactions in situ with high specificity and sensitivity.

• Mutational analysis coupling: Combine antibody-based detection with expression of PARD6B mutants (K19A to disrupt aPKC binding, ΔPro136 to disrupt CDC42 binding, or M235W to disrupt Lgl binding) to validate interaction specificity .

• Sequential co-IP: For complex multi-protein assemblies, perform sequential co-IPs to identify hierarchical assembly patterns of the PAR complex components.

What considerations are important when designing PARD6B knockdown validation experiments?

When validating PARD6B knockdown effects, proper antibody-based confirmation is essential:

• Cross-validation with multiple antibodies: Use at least two different antibodies targeting different PARD6B epitopes to confirm knockdown efficiency at the protein level .

• Knockdown controls: Include appropriate controls:

  • Non-targeting siRNA/shRNA controls

  • Rescue experiments with siRNA/shRNA-resistant PARD6B constructs

  • Positive controls targeting proteins with well-established knockdown phenotypes

• Quantification methods: Employ quantitative Western blotting with validated PARD6B antibodies (1:500-1:2000 dilution) and appropriate loading controls to measure knockdown efficiency .

• Phenotypic assays: Following knockdown verification, use PARD6B antibodies in functional assays to assess consequences:

  • Tight junction formation using TJP1 (ZO-1) co-staining

  • Cell polarity using aPKC localization

  • CDX2 expression in developmental contexts

• Cell-type specificity: Different cell types may show varying knockdown efficiencies and phenotypes. For example, knockdown in MCF7 cells results in loss of TJ assembly and membrane localization of aPKC .

What quality control measures should researchers implement when working with PARD6B antibodies?

To ensure reproducible and reliable results with PARD6B antibodies:

• Antibody validation panel: Implement a comprehensive validation strategy:

• Lot-to-lot consistency: Test new antibody lots against previous lots to ensure consistent performance, particularly for long-term studies.

• Application-specific controls: Include appropriate positive and negative controls for each application:

  • For IHC: mouse kidney tissue and human pancreas tissue as positive controls

  • For IF: HepG2 and MCF-7 cells as positive controls

  • For WB: HeLa, HEK-293, human placenta tissue, and PC-3 cell lysates as positive controls

• Proper antibody storage: Store antibodies according to manufacturer recommendations (typically at -20°C in small aliquots to avoid freeze-thaw cycles) to maintain reactivity .

How can researchers distinguish between true PARD6B signals and non-specific binding in complex experimental systems?

Distinguishing specific from non-specific signals requires rigorous controls:

• Multiple antibody validation: Use antibodies from different sources targeting different epitopes of PARD6B. Consistent patterns across antibodies suggest specific detection.

• Blocking peptide competition: Pre-incubate the antibody with the specific immunogen peptide used to generate it. The peptide will compete for antibody binding, and specific signals should be significantly reduced or eliminated.

• Genetic approaches: Use PARD6B knockdown or knockout systems as negative controls. For instance, microinjecting RNA interference constructs targeting PARD6B into zygotes provides a powerful negative control for antibody specificity in developmental studies .

• Isotype controls: Include appropriate isotype controls matched to the PARD6B antibody (e.g., rabbit IgG for rabbit polyclonal antibodies or mouse IgG2a for monoclonal antibodies like sc-166405) .

• Signal quantification: Always quantify signals relative to appropriate controls, and use statistical analysis to determine significance of observed differences.

• Alternative detection methods: Corroborate antibody-based results with non-antibody methods such as mRNA expression analysis or tagged protein expression.

What are the advantages and limitations of polyclonal versus monoclonal PARD6B antibodies for different research applications?

Understanding the strengths and limitations of different antibody types is crucial for experimental design:

For critical experiments, consider using both types to leverage their complementary advantages and provide validation through concordant results .

How can PARD6B antibodies be utilized in emerging single-cell analysis technologies?

As single-cell technologies advance, PARD6B antibodies can be integrated into these platforms:

• Mass cytometry (CyTOF): Metal-conjugated PARD6B antibodies can be incorporated into CyTOF panels to analyze PARD6B expression alongside dozens of other markers at single-cell resolution, particularly useful for heterogeneous samples like developmental systems or tumors.

• Single-cell Western blotting: Validated PARD6B antibodies can be adapted for microfluidic single-cell Western blot protocols to analyze protein expression variability across individual cells.

• Proximity extension assays: PARD6B antibody pairs can be developed for highly sensitive detection of protein expression in limited samples through oligonucleotide-conjugated antibody approaches.

• Spatial transcriptomics correlation: Combine PARD6B immunofluorescence with spatial transcriptomics to correlate protein localization with gene expression patterns at tissue and subcellular levels.

• Live-cell imaging: Develop non-perturbing nanobodies against PARD6B for live-cell tracking of dynamic protein localization and interaction during processes like cell division and polarization.

What methodological advances could improve detection of post-translational modifications of PARD6B?

Post-translational modifications (PTMs) often regulate PARD6B function, but their detection remains challenging:

• Phospho-specific antibodies: Develop antibodies against specific phosphorylation sites that regulate PARD6B function, particularly those mediating interaction with aPKC or affecting PDZ domain binding.

• PTM-IP strategies: Use general PARD6B antibodies for immunoprecipitation followed by mass spectrometry analysis to identify novel PTMs and their dynamics during cellular processes.

• Phos-tag gel electrophoresis: Combine validated PARD6B antibodies with Phos-tag SDS-PAGE to separate and detect phosphorylated forms of PARD6B without requiring phospho-specific antibodies.

• Proximity ligation approaches: Develop protocols using PARD6B antibodies in conjunction with antibodies against specific PTMs (phospho, ubiquitin, SUMO) to visualize modified populations in situ.

• Sequential IP strategy: Perform sequential immunoprecipitation first with PTM-specific antibodies followed by PARD6B detection, or vice versa, to enrich for modified forms of the protein.

These methodological advances would significantly enhance our understanding of how PARD6B activity is regulated in different cellular contexts and could reveal new therapeutic opportunities in diseases where PARD6B function is dysregulated.