PARD6A Antibody, HRP conjugated

What is PARD6A and what are its main cellular functions?

PARD6A (also known as PAR-6A, PAR6alpha, or PAR6C) is a scaffold protein that functions as a critical component of the PAR complex, which establishes and maintains cell polarity. It assembles with atypical protein kinase C (aPKC) and the small Rho-GTPase Cdc42 to define cell polarity and regulate membrane trafficking . PARD6A is primarily localized to cell junctions, cell membranes, cell projections, cytoplasmic ruffles, and tight junctions .

At the molecular level, PARD6A contains PDZ domains that enable protein-protein interactions with other polarity regulators like Par3. Research has shown that one Par3 protein can potentially bind to two Par6 proteins, which may serve as the foundation for polarity-generating protein scaffolds in cells . This architecture allows for the precise spatial organization required for establishing cellular asymmetry.

Dysregulation of PARD6A has been implicated in various pathological conditions, including cancer progression and neurological disorders, making it an important target for research investigations .

What are the key characteristics of HRP-conjugated PARD6A antibody?

The HRP-conjugated PARD6A antibody is a rabbit polyclonal antibody directly conjugated with horseradish peroxidase (HRP) for enhanced detection sensitivity. This conjugation eliminates the need for secondary antibodies in experimental workflows. The antibody is typically:

• Generated in rabbits immunized with recombinant human PARD6A protein (amino acids 248-344)

• Purified using Protein G affinity chromatography with >95% purity

• Stored in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

• Maintained at -20°C or -80°C to preserve activity, avoiding repeated freeze-thaw cycles

• Reactive primarily with human PARD6A, though cross-reactivity with other species may occur depending on sequence conservation

The HRP conjugation provides direct enzymatic activity for colorimetric or chemiluminescent detection in applications such as ELISA and Western blotting.

How do the different PAR6 isoforms (PARD6A, PARD6B, PARD6D) differ functionally?

In mammals, there are three closely related isoforms of PAR6: PARD6A, PARD6B, and PARD6D. While they share structural similarities, they exhibit distinct functional specializations:

Research using gene knockouts or depletion experiments has demonstrated that these isoforms are not entirely redundant. For instance, in studies of apical endosome function, depletion of PARD6B affected transcytosis and apical recycling, but these functions could not be rescued by overexpression of either PARD6A or PARD6D . This indicates that despite structural similarities, each isoform likely mediates distinct aspects of cellular polarity regulation.

How should researchers optimize Western blot protocols for PARD6A detection using HRP-conjugated antibodies?

Optimizing Western blot protocols for PARD6A detection requires attention to several critical parameters:

Sample Preparation:

• Include protease inhibitors in lysis buffers to prevent degradation of PARD6A

• Use phosphatase inhibitors if studying phosphorylated forms of PARD6A or associated proteins

• For membrane-associated PARD6A, ensure lysis buffers contain appropriate detergents

Protocol Optimization:

• Dilution Range: Begin with a 1:500 to 1:2000 dilution as recommended for PARD6A antibodies

• Blocking Solution: 5% non-fat dry milk or BSA in TBST; test both if experiencing background issues

• Incubation Conditions: Primary antibody incubation at 4°C overnight often yields better results than shorter incubations at room temperature

• Washing Steps: Implement stringent washing with TBST (at least 3-5 washes, 5-10 minutes each)

• Detection System: Select ECL substrate sensitivity based on expected protein abundance

Important Considerations:

• The calculated molecular weight of PARD6A is 37 kDa, but it typically appears at ~47 kDa on SDS-PAGE due to post-translational modifications

• Multiple bands may indicate different isoforms, degradation products, or post-translationally modified variants

• Always include positive controls like extracts from SW620, HT-29, Jurkat, U-251MG, or Raji cell lines

• When analyzing PARD6A in specialized contexts (e.g., polarity complexes), consider co-immunoprecipitation followed by Western blotting

What are the optimal conditions for immunoprecipitation studies investigating PARD6A interactions with polarity complex proteins?

Immunoprecipitation (IP) is a valuable technique for studying PARD6A interactions with other components of polarity complexes such as Cdc42, aPKC, and Par3. Optimizing IP conditions requires consideration of several factors:

Buffer Composition:

• Use mild lysis buffers (e.g., 1% NP-40 or Triton X-100) to preserve protein-protein interactions

• Include protease inhibitors to prevent degradation

• Consider phosphatase inhibitors if studying phosphorylation-dependent interactions

• Adjust salt concentration (150-300 mM NaCl) depending on interaction strength

Protocol Recommendations:

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

• For co-IP of PARD6A with Cdc42, consider using GST-JFC1D1 as a pull-down method for active Rab8, which has been shown to work in similar polarity complex studies

• When studying PARD6A interaction with GRAF1 and Cdc42, the APEX-tagged proximity labeling approach has proven effective for identifying neighboring proteins in the complex

• For weak or transient interactions, consider using crosslinking reagents before lysis

Data Interpretation:

• Always include appropriate negative controls (non-specific IgG, lysates from cells with PARD6A knockdown)

• Validate interactions using reverse co-IP (immunoprecipitate the binding partner and blot for PARD6A)

• Consider proximity ligation assays as a complementary approach to confirm interactions in intact cells

Research has demonstrated that PARD6A interactions can be detected through multiple approaches, including the APEX-tagged proximity labeling system, which successfully identified interactions between GRAF1, Cdc42, Rab8, EHD1, and MICAL-L1 in polarized cells .

How can researchers troubleshoot non-specific binding when using PARD6A antibody?

Non-specific binding is a common challenge when working with antibodies, including those against PARD6A. Here are comprehensive troubleshooting strategies:

Sources of Non-Specific Binding:

• Insufficient blocking

• Suboptimal antibody dilution

• Cross-reactivity with similar epitopes

• Sample overloading

• Inadequate washing

Systematic Troubleshooting Approach:

Advanced Validation Techniques:

• Peptide competition assay: Pre-incubate antibody with the immunizing peptide before application

• Multiple antibody validation: Test different antibodies targeting distinct epitopes of PARD6A

• Immunodepletion: Sequential probing of the same lysate with the same antibody should show signal reduction

When interpreting results, remember that PARD6A is observed at approximately 47 kDa despite a calculated molecular weight of 37 kDa , which could be due to post-translational modifications or the presence of protein complexes.

How can PARD6A antibody be used to study its role in establishing and maintaining cell polarity?

Investigating PARD6A's role in cell polarity requires sophisticated experimental approaches. The HRP-conjugated PARD6A antibody can be employed in several advanced experimental designs:

Polarized Epithelial Model Systems:

• Grow epithelial cells (MDCK, Caco-2) on Transwell filters to establish apical-basolateral polarity

• Use immunofluorescence with PARD6A antibody to track its localization during polarization

• Correlate PARD6A distribution with tight junction markers (ZO-1, occludin) and other polarity proteins (Par3, aPKC)

Asymmetric Cell Division Models:

• Track PARD6A localization during mitosis in stem cell populations

• Correlate PARD6A distribution with asymmetric fate determinants

• Analyze how perturbation of PARD6A affects daughter cell fate specification

Polarity Complex Formation Analysis:
Research has shown that Par3 protein can bind to two Par6 proteins, potentially forming the basis for polarity-generating protein scaffolds . This can be investigated through:

• Co-immunoprecipitation of PARD6A with Par3 followed by Western blotting

• Proximity ligation assays to visualize PARD6A-Par3 interactions in situ

• FRET or BRET analyses to measure direct protein-protein interactions in live cells

Membrane Dynamics Studies:
PARD6A has been linked to membrane trafficking processes. Studies have shown that depletion of GRAF1, which interacts with components of the polarity complex, affects Rab8 activity and alters the localization of phosphorylated Par6 from uniform membrane distribution to asymmetric redistribution . Researchers can investigate:

• The co-localization of PARD6A with membrane trafficking markers

• The effect of PARD6A depletion on endocytic recycling

• How PARD6A phosphorylation status affects its membrane recruitment

What experimental approaches can distinguish the functions of different PAR6 isoforms?

Distinguishing the functions of PARD6A, PARD6B, and PARD6D requires careful experimental design:

Isoform-Specific Depletion and Rescue:
Research has demonstrated that depletion of PARD6B affects apical endosome function, and this phenotype cannot be rescued by overexpression of PARD6A or PARD6D . Similar approaches can be used to identify other isoform-specific functions:

• Use isoform-specific siRNA/shRNA to deplete individual PAR6 proteins

• Perform rescue experiments with expression constructs for each isoform

• Measure functional readouts relevant to polarity (barrier function, protein trafficking, etc.)

Domain Swap Experiments:

• Create chimeric proteins by swapping domains between PARD6A, PARD6B, and PARD6D

• Express these in cells depleted of endogenous PAR6 proteins

• Identify which domains confer isoform-specific functions

Proteomic Analysis of Isoform-Specific Interactomes:

• Perform immunoprecipitation of each PAR6 isoform individually

• Analyze binding partners by mass spectrometry

• Compare interactomes to identify unique and shared interaction partners

Tissue-Specific Expression Analysis:

• Use isoform-specific antibodies to map expression patterns across tissues

• Correlate expression with tissue-specific polarity phenomena

• Investigate how tissue environments might regulate isoform expression

Experimental Data Example:
In studies of polarized epithelial cells, depletion of PARD6B resulted in defects in transcytosis and apical recycling. When researchers attempted to rescue these defects by overexpressing myc-tagged PARD6A or PARD6D, they found that neither isoform could restore normal function, while reintroduction of myc-tagged PARD6B successfully rescued the phenotype . This demonstrates that despite structural similarities, PARD6B has unique functional properties in apical endosome regulation that cannot be compensated by other isoforms.

How can researchers use PARD6A antibody to investigate its roles in disease models?

PARD6A dysregulation has been implicated in various pathological conditions, including cancer and neurological disorders . HRP-conjugated PARD6A antibody can be instrumental in investigating disease mechanisms:

Cancer Research Applications:

• Tumor Tissue Array Analysis:

  • Compare PARD6A expression levels and localization between normal and tumor tissues

  • Correlate expression patterns with clinical parameters and patient outcomes

  • Identify potential biomarker value

• Epithelial-to-Mesenchymal Transition (EMT) Studies:

  • Track PARD6A redistribution during EMT induction

  • Correlate changes with loss of cell polarity and increased invasiveness

  • Investigate PARD6A interaction with EMT-related signaling pathways

• 3D Tumor Organoid Models:

  • Analyze PARD6A localization in 3D cultures that better recapitulate tumor architecture

  • Study how PARD6A affects organoid formation and polarity establishment

  • Test how targeting PARD6A impacts tumor organoid growth and invasion

Neurological Disorder Models:

• Investigate PARD6A distribution in neuronal cultures from disease models

• Correlate PARD6A localization with neuronal polarity defects

• Examine how disease-associated mutations affect PARD6A function

Experimental Approach Considerations:

• Use multiple detection methods (Western blot, immunofluorescence, IHC) for comprehensive assessment

• Include appropriate positive controls (such as SW620, HT-29, or Jurkat cell lines)

• Validate findings with genetic manipulation (knockdown/overexpression)

• Consider combined staining with other markers to assess contextual changes in polarity complex formation

For instance, in cancer studies, researchers could investigate how PARD6A interacts with the PAR complex and whether alterations in this interaction contribute to loss of cell polarity and increased invasiveness. The HRP-conjugated antibody would be particularly useful for sensitive detection in tissue samples where protein expression might be variable.

What are the optimal fixation and immunostaining protocols for detecting PARD6A in different cell types?

Optimizing fixation and immunostaining protocols is crucial for accurate detection of PARD6A, which is found in multiple cellular compartments including cell junctions, membrane, projections, cytoplasm, and tight junctions :

Fixation Method Comparison:

Cell Type-Specific Considerations:

Different cell types may require tailored approaches:

• Epithelial Cells (MDCK, Caco-2):

  • Grow on Transwell filters for proper polarization

  • Fix from both apical and basolateral compartments

  • Consider z-stack confocal imaging to properly resolve apical vs. basolateral staining

• Neuronal Cells:

  • Gentler fixation protocols to preserve delicate processes

  • Longer permeabilization time for dense cytoskeletal networks

  • Consider co-staining with axonal/dendritic markers to assess polarized distribution

• Cancer Cell Lines:

  • Compare fixation methods, as cancer cells may have altered membrane properties

  • For SW620, HT-29, and Jurkat cells (known positive controls) , PFA fixation with Triton X-100 permeabilization generally works well

Step-by-Step Optimized Protocol:

• Wash cells in PBS (2-3 times)

• Fix with 4% PFA for 15-20 minutes at room temperature

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

• Permeabilize with 0.2% Triton X-100 in PBS for 5-10 minutes

• Block with 5% normal serum (matching secondary antibody host) in PBS for 1 hour

• Incubate with PARD6A antibody (1:100-1:500 dilution) overnight at 4°C

• Wash with PBS + 0.1% Tween-20 (3 times, 5 minutes each)

• For HRP-conjugated antibodies: Develop using appropriate substrate
  For unconjugated antibodies: Incubate with secondary antibody (1:500) for 1 hour at room temperature

• Counterstain nuclei with DAPI if desired

• Mount and analyze

Troubleshooting High Background:

• Increase antibody dilution (1:500 - 1:1000)

• Extend blocking time to 2 hours

• Add 0.1% BSA to antibody dilution buffer

• Perform additional washing steps

How can researchers quantitatively analyze PARD6A expression and distribution in complex tissues?

Quantitative analysis of PARD6A in complex tissues requires sophisticated approaches to address heterogeneity and spatial organization:

Sample Preparation Considerations:

• Use consistent fixation protocols across all samples

• Consider antigen retrieval optimization for tissue sections (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Include positive control tissues with known PARD6A expression

• Process experimental and control samples in parallel to minimize batch effects

Quantification Methodologies:

Digital Image Analysis Workflow:

• Acquire high-resolution images using standardized microscope settings

• Apply flat-field correction to normalize illumination

• Define regions of interest (cellular compartments, tissue areas)

• Segment images to identify positive staining

• Extract quantitative parameters:

  • Mean fluorescence/staining intensity

  • Percentage of positive cells

  • Subcellular distribution patterns

  • Co-localization coefficients with other markers

Advanced Analysis Approaches:

• Spatial Statistics: Analyze the clustering or dispersion of PARD6A staining relative to tissue landmarks

• Multi-parameter Analysis: Correlate PARD6A with other polarity markers (aPKC, Par3) and functional outcomes

• Machine Learning Classification: Train algorithms to recognize patterns of PARD6A distribution associated with specific phenotypes

Research has demonstrated that PARD6A localization can be dynamic and context-dependent. For example, in polarized cells, phosphorylated Par6 is normally detected along the entire membrane, but GRAF1 depletion causes asymmetric redistribution . These subtle changes in distribution pattern require careful quantitative analysis rather than simple positive/negative assessment.

What controls are essential when using PARD6A antibody for critical experiments?

Rigorous controls are essential for ensuring the reliability and interpretability of experiments using PARD6A antibody:

Antibody Validation Controls:

• Positive Controls:

  • Cell lines with known PARD6A expression (SW620, HT-29, Jurkat, U-251MG, Raji)

  • Mouse testis tissue, which has demonstrated PARD6A expression

  • Recombinant PARD6A protein (for Western blot)

• Negative Controls:

  • Primary antibody omission

  • Non-specific IgG from the same species (rabbit for most PARD6A antibodies)

  • Cells with PARD6A knockdown via siRNA/shRNA

  • If available, tissues/cells from PARD6A knockout models

• Specificity Controls:

  • Peptide competition/blocking with the immunizing peptide

  • Testing multiple antibodies against different PARD6A epitopes

  • Western blot showing a single band at expected molecular weight (~47 kDa)

Experimental Design Controls:

Critical Controls for Specific Applications:

• Co-immunoprecipitation:

  • Input lysate control

  • IgG pull-down control

  • Reverse co-IP (immunoprecipitate binding partner, detect PARD6A)

• Immunofluorescence:

  • Single-color controls for multi-channel imaging

  • Background autofluorescence control

  • Co-staining with markers of known cellular compartments

• Functional Studies:

  • Rescue experiments with wild-type PARD6A after knockdown

  • Comparison with other PAR6 isoforms (PARD6B, PARD6D)

  • Parallel analysis of known PARD6A downstream targets

Data Interpretation Considerations:

How can PARD6A antibody be used to investigate its involvement in cancer progression mechanisms?

PARD6A dysregulation has been implicated in cancer development and progression . Using HRP-conjugated PARD6A antibody, researchers can investigate several key aspects of cancer biology:

Mechanisms of Cancer Cell Polarity Loss:

• Compare PARD6A localization between normal and transformed epithelial cells

• Investigate how oncogenic signaling alters PARD6A distribution and function

• Determine whether PARD6A mislocalization correlates with loss of tissue architecture

PARD6A in Cancer Cell Migration and Invasion:
Research has shown that proper polarity protein function is essential for controlled cell migration. Dysregulation of polarity complexes can contribute to enhanced invasiveness. Researchers can:

• Track PARD6A distribution during cancer cell migration using live-cell imaging

• Correlate PARD6A localization patterns with invasive capacity

• Determine how manipulation of PARD6A levels affects migration/invasion in 3D matrices

Therapeutic Targeting Opportunities:

• Use PARD6A antibody to assess target engagement in drug development

• Investigate how restoring PARD6A function affects cancer cell phenotypes

• Explore PARD6A as a biomarker for treatment response

Experimental Model Recommendations:

Research Questions to Explore:

• Does PARD6A expression correlate with clinical outcomes in specific cancer types?

• How does PARD6A interact with known oncogenes or tumor suppressors?

• Can targeting PARD6A or its interactions restore normal cell polarity and reduce malignant behaviors?

What are the emerging techniques for studying PARD6A dynamics in live cells?

Emerging techniques offer new opportunities for studying PARD6A dynamics with unprecedented resolution:

Advanced Live Imaging Approaches:

• Fluorescent Protein Tagging:

  • Generate PARD6A-GFP/RFP fusion constructs

  • Observe real-time recruitment to polarity complexes

  • Track movement between cellular compartments

• FRAP (Fluorescence Recovery After Photobleaching):

  • Measure PARD6A mobility in different cellular domains

  • Determine if disease states alter protein dynamics

  • Identify factors affecting PARD6A retention at cell junctions

• Single-Molecule Tracking:

  • Visualize individual PARD6A molecules

  • Characterize diffusion rates and confinement zones

  • Identify transient interactions with other polarity proteins

Proximity-Based Detection Methods:

• APEX Proximity Labeling:
  Research has shown that APEX-tagged GRAF1 successfully identified nearby proteins including Rab8, Cdc42, EHD1, and MICAL-L1 in polarized cells . Similar approaches could be applied specifically to PARD6A:

  • Generate PARD6A-APEX fusion proteins

  • Identify proteins in close proximity through biotinylation

  • Map the PARD6A "neighborhood" in different cellular contexts

• BioID Approach:

  • Express PARD6A-BioID fusion in cells

  • Identify biotinylated proximity partners

  • Compare interactome changes during polarization

• Split-GFP Complementation:

  • Create PARD6A and potential partner split-GFP fusions

  • Visualize interactions through reconstituted fluorescence

  • Monitor interaction dynamics in real-time

Optogenetic Control:

• Develop light-controlled PARD6A recruitment systems

• Study consequences of acute PARD6A mislocalization

• Determine minimum requirements for polarity complex function

Technical Considerations:

• Ensure that tags don't interfere with PARD6A function

• Validate fusion protein localization against endogenous PARD6A

• Consider photobleaching, phototoxicity, and expression levels

• Include appropriate controls (non-interacting proteins, mutant versions)

How can researchers investigate the cross-talk between PARD6A and other signaling pathways?

PARD6A functions within a complex network of signaling pathways. Investigating these interactions requires sophisticated experimental approaches:

PARD6A Interactions with Rho GTPase Signaling:
Research has demonstrated connections between PARD6A and Cdc42, a small Rho-GTPase that defines cell polarity and regulates membrane trafficking .

• GTPase Activity Assays:

  • Pull-down active GTPases using GST-tagged binding domains

  • Determine how PARD6A manipulation affects GTPase activation

  • Connect GTPase activity to downstream polarity phenotypes

• Effector Pathway Analysis:

  • Monitor phosphorylation of downstream targets (PAK, LIMK)

  • Assess cytoskeletal organization following PARD6A manipulation

  • Determine how GTPase inhibitors affect PARD6A localization

PARD6A and aPKC Signaling:
PARD6A forms a complex with aPKC that regulates numerous cellular processes :

• Phosphorylation Studies:

  • Analyze aPKC substrate phosphorylation following PARD6A modulation

  • Investigate PARD6A phosphorylation status using phospho-specific antibodies

  • Determine how phosphatase inhibitors affect PARD6A function

• Polarity Complex Assembly Analysis:

  • Track recruitment sequence of polarity proteins during polarization

  • Investigate how disrupting one component affects others

  • Research has shown that Par6 phosphorylation by aPKC affects its distribution along the membrane

Experimental Approaches to Study Cross-Talk: