PARD6A Antibody, Biotin conjugated

What is PARD6A and what cellular functions does it regulate?

PARD6A (par-6 family cell polarity regulator alpha) is a 346-amino acid protein belonging to the PAR6 family. It functions primarily as a scaffolding protein involved in establishing and maintaining cell polarity across diverse cellular contexts. PARD6A localizes predominantly to the cytoplasm and membrane regions where it forms a conserved polarity complex with PAR3 and atypical protein kinase C (aPKC). This complex plays essential roles in asymmetric cell division, epithelial tight junction formation, directional cell migration, and neuronal differentiation. The protein contains multiple domains facilitating protein-protein interactions, including a PB1 domain that mediates heterodimerization with aPKC, a semi-CRIB domain that binds activated CDC42/Rac1 GTPases, and a PDZ domain that interacts with PAR3 and other junction proteins. These interactions collectively establish and maintain apicobasal polarity in epithelial cells through regulation of cytoskeletal dynamics and membrane protein trafficking .

How does biotin conjugation enhance PARD6A antibody functionality?

Biotin conjugation to PARD6A antibodies creates a versatile research tool by leveraging the exceptionally high affinity interaction between biotin and streptavidin/avidin (Kd ≈ 10^-15 M). This conjugation enables signal amplification through multiple detection strategies without altering the antibody's target specificity. The small biotin molecule (244 Da) minimally affects antibody binding kinetics while providing a robust handle for detection. In multi-step labeling protocols, biotin-conjugated antibodies allow for sequential application of detection reagents, reducing background and cross-reactivity issues common in direct fluorophore conjugates. Additionally, the biotin-streptavidin system functions effectively across a wide pH range (pH 4-10) and remains stable under harsh washing conditions, enabling stringent protocol optimization. For PARD6A detection specifically, biotin conjugation helps overcome the challenges of detecting this relatively low-abundance scaffolding protein, particularly in tissues with high autofluorescence or when examining its redistribution during polarity establishment .

What are the key differences between polyclonal and monoclonal PARD6A antibodies for research applications?

Polyclonal and monoclonal PARD6A antibodies offer distinct advantages depending on experimental requirements:

For studies focusing on PARD6A localization dynamics during polarization events, monoclonal antibodies often provide more consistent results when comparing multiple experimental conditions or timepoints. Conversely, polyclonal antibodies may be advantageous for initial detection in tissues with low PARD6A expression or when attempting to capture all forms of the protein regardless of post-translational modifications .

What are the optimal sample preparation protocols for PARD6A detection in different cell types?

Sample preparation protocols require optimization based on the subcellular localization of PARD6A in different cell types:

For epithelial cells (e.g., MDCK, Caco-2):

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

• For membrane-associated PARD6A detection, avoid methanol fixation which can disrupt membrane structures

• Include a mild permeabilization step (0.2% Triton X-100 for 10 minutes) to access cytoplasmic and membrane-associated protein

• Block with 5% BSA containing 0.1% saponin to maintain tight junction integrity while allowing antibody access

For neuronal cultures:

• Use a sequential fixation protocol (4% paraformaldehyde followed by -20°C methanol) for detecting PARD6A in neuronal growth cones

• Extend blocking time to 2 hours at room temperature with 10% normal goat serum to reduce non-specific binding

• Include 0.05% Tween-20 in all washing steps to reduce background

For tissue sections:

• Perform antigen retrieval using sodium citrate buffer (pH 6.0) at 95°C for 20 minutes

• For paraffin sections, complete deparaffinization is critical as incomplete removal of paraffin dramatically reduces PARD6A detection sensitivity

• For frozen sections, a post-fixation step with 2% paraformaldehyde helps maintain tissue morphology without reducing antibody accessibility

Critical parameter: Regardless of sample type, avoid harsh detergents and excessive permeabilization as these can disrupt the protein complexes PARD6A forms with PAR3 and aPKC, potentially resulting in false-negative results .

How should researchers optimize dilution ratios for biotin-conjugated PARD6A antibodies across different applications?

Optimization of dilution ratios for biotin-conjugated PARD6A antibodies should follow application-specific strategies:

Titration should be performed for each new experimental system and antibody lot. For PARD6A specifically, researchers should validate optimal dilutions using both positive controls (cell lines with known PARD6A expression like MDCK or MCF-7 cells) and negative controls (PARD6A-knockdown cells or tissues). The biotin-conjugated antibody may require 25-50% less concentrated dilutions compared to unconjugated versions due to the signal amplification provided by the detection system .

What detection systems work most effectively with biotin-conjugated PARD6A antibodies?

Multiple detection systems can be employed with biotin-conjugated PARD6A antibodies, each offering distinct advantages:

For studies examining PARD6A co-localization with other polarity proteins, fluorescent detection systems using spectrally distinct fluorophores (like Streptavidin-Alexa Fluor® 488 for PARD6A and directly labeled antibodies with longer wavelength fluorophores for interacting partners) provide optimal results. When quantitative analysis is required, standardizing exposure settings using calibration beads helps ensure comparable signal intensity across experiments .

How can PARD6A antibodies be effectively used to study protein-protein interactions within polarity complexes?

PARD6A functions primarily through protein-protein interactions within polarity complexes, and biotin-conjugated antibodies can be leveraged for multiple interaction analysis approaches:

• Proximity Ligation Assay (PLA):

  • Combine biotin-conjugated PARD6A antibody with unconjugated antibodies against potential interaction partners (e.g., PAR3, aPKC, CDC42)

  • Use streptavidin-oligonucleotide and secondary antibody-oligonucleotide conjugates as PLA probes

  • Each detected fluorescent spot represents a molecular interaction (<40nm proximity)

  • Quantification of PLA signals allows comparative analysis of interaction dynamics during polarization events

• Sequential Co-Immunoprecipitation:

  • Perform first immunoprecipitation using biotin-conjugated PARD6A antibody captured on streptavidin-magnetic beads

  • Elute complexes under mild conditions preserving protein-protein interactions

  • Subject eluate to second immunoprecipitation with antibodies against suspected interaction partners

  • This approach identifies stable subcomplexes within the larger polarity network

• BiFC (Bimolecular Fluorescence Complementation) Validation:

  • After identifying potential interactions by co-immunoprecipitation, validate using BiFC

  • Biotin-conjugated PARD6A antibodies can confirm proper expression and localization of fusion proteins

The choice of method depends on whether researchers are investigating stable or transient interactions, and whether spatial information about the interaction site is required. For membrane-associated PARD6A complexes, detergent selection during sample preparation is critical - CHAPS or digitonin (0.5-1%) generally preserve interactions better than stronger detergents like Triton X-100 .

What are common causes of false positives/negatives when using PARD6A antibodies, and how can researchers mitigate these issues?

When working with PARD6A antibodies, several technical challenges can lead to misleading results:

To systematically address these issues, researchers should implement a validation pipeline including: (1) parallel analysis with multiple PARD6A antibodies, (2) validation in systems with modulated PARD6A expression, (3) pre-absorption controls, and (4) careful selection of positive and negative control tissues or cell lines with known PARD6A expression patterns .

How can researchers effectively use PARD6A antibodies to study polarization dynamics during epithelial morphogenesis?

Studying polarization dynamics during epithelial morphogenesis requires specialized approaches:

• Time-resolved immunofluorescence:

  • Fix 3D epithelial cultures (e.g., MDCK cysts) at defined stages of morphogenesis (2h, 4h, 8h, 12h, 24h, 48h, 72h)

  • Process for immunofluorescence using biotin-conjugated PARD6A antibody and markers for adherens junctions (E-cadherin), tight junctions (ZO-1), and apical membrane (gp135/podocalyxin)

  • Analyze changes in PARD6A localization relative to these markers using confocal microscopy

  • Quantify co-localization coefficients (Pearson's or Manders') to track temporal changes in PARD6A association with different membrane domains

• Calcium-switch experiments:

  • Grow epithelial monolayers in low-calcium medium to disrupt cell-cell adhesion

  • Restore normal calcium levels and fix cells at defined timepoints

  • Immunostain for PARD6A and junctional markers

  • This approach allows synchronized analysis of polarity complex assembly

• FRAP (Fluorescence Recovery After Photobleaching) validation:

  • After immunofluorescence studies identify key transition points, validate with live-cell FRAP

  • Express fluorescently-tagged PARD6A and measure protein dynamics

  • Use antibody staining to confirm that tagged protein localizes similarly to endogenous protein

In these studies, biotin-conjugated PARD6A antibodies offer advantages for multiplexed staining, allowing simultaneous visualization of multiple polarity and junctional proteins. For optimal results in 3D cultures, researchers should extend primary antibody incubation times (24-48h at 4°C) and use detection reagents conjugated to bright, photostable fluorophores like Alexa Fluor 647 to minimize interference from autofluorescence .

What considerations are important when using PARD6A antibodies for quantitative analysis of protein expression?

Quantitative analysis of PARD6A expression requires careful experimental design and standardization:

For Western blot quantification, researchers should optimize protein extraction protocols to efficiently solubilize membrane-associated PARD6A (using buffers containing 0.5% NP-40 or 1% Triton X-100) and include phosphatase inhibitors to preserve post-translational modifications that may affect antibody recognition. When analyzing PARD6A redistribution during polarization, reporting both absolute expression and relative distribution (membrane/cytosolic ratio) provides the most complete picture of polarization dynamics .

How can PARD6A antibodies be utilized in cancer research studying cell polarity dysregulation?

PARD6A dysregulation has been implicated in multiple cancer types, particularly breast, colorectal, and prostate cancers, where alterations in cell polarity contribute to tumor progression. Biotin-conjugated PARD6A antibodies offer several advantages in cancer research applications:

• Tissue Microarray (TMA) Analysis:

  • TMAs containing multiple patient samples can be stained with biotin-conjugated PARD6A antibodies

  • Streptavidin-HRP detection systems provide consistent staining across large sample numbers

  • Quantitative image analysis can correlate PARD6A expression or mislocalization with clinical parameters

• Epithelial-Mesenchymal Transition (EMT) Studies:

  • PARD6A relocalization is an early marker of polarity loss during EMT

  • Dual immunofluorescence with PARD6A and EMT markers (E-cadherin, vimentin, Snail) tracks transition states

  • Biotin-conjugated antibodies allow flexible secondary detection strategies for multiplexed imaging

• Metastatic Potential Assessment:

  • Compare PARD6A localization patterns between primary tumors and metastatic sites

  • Correlate patterns with invasive capacity in transwell or organotypic culture models

  • Developed scoring system example:

For cancer studies, standardized IHC protocols are critical for reliable comparison across patient cohorts. Automated staining platforms provide the most consistent results for large-scale studies, while manual protocols may be preferred for optimizing detection in specific tumor types .

What approaches can researchers use to study post-translational modifications of PARD6A?

PARD6A function is regulated through multiple post-translational modifications (PTMs), with phosphorylation and ubiquitination being particularly important for controlling protein stability and complex formation:

• Phosphorylation Analysis:

  • Use phosphorylation-state specific antibodies alongside general PARD6A antibodies

  • Compare detection patterns under different cellular conditions

  • Confirm specificity with lambda phosphatase treatment controls

  • Employ 2D gel electrophoresis followed by Western blotting to resolve different phosphorylated species

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Perform immunoprecipitation using biotin-conjugated PARD6A antibodies

  • Analyze precipitated complexes by mass spectrometry to identify:

    • Phosphorylation sites (enrichment with TiO₂ or IMAC)

    • Ubiquitination sites (after tryptic digestion, look for GG remnants)

    • Interaction partners that may regulate modifications

• Assessing Modification Dynamics:

  • Treat cells with pathway inhibitors targeting known PARD6A-modifying kinases (PKC, PAR1)

  • Monitor changes in PARD6A localization and complex formation

  • Time-course experiments following polarization stimuli

When studying PARD6A modifications, sample preparation should include appropriate inhibitors (phosphatase inhibitors for phosphorylation studies, deubiquitinase inhibitors like N-ethylmaleimide for ubiquitination studies) to preserve the modification state during extraction and analysis .

How can researchers effectively use PARD6A antibodies in neuronal polarity studies?

PARD6A plays crucial roles in neuronal development, particularly in axon specification and dendritic spine morphogenesis. Specialized approaches for neuronal studies include:

• Developmental Timeline Analysis:

  • Prepare primary neuronal cultures from embryonic rodent cortex or hippocampus

  • Fix and immunostain cultures at key developmental stages (6h, 12h, 24h, 3DIV, 7DIV, 14DIV)

  • Use biotin-conjugated PARD6A antibody with neuronal markers (Tau-1 for axons, MAP2 for dendrites)

  • Quantify PARD6A enrichment in developing axonal growth cones versus dendritic growth cones

• Subcellular Localization in Mature Neurons:

  • For dendritic spine analysis, use super-resolution microscopy (STED, STORM) after PARD6A immunostaining

  • Categorize spines morphologically (mushroom, thin, stubby) and quantify PARD6A distribution

  • Co-stain with synaptic markers (PSD-95, synaptophysin) to assess synaptic localization

• Optogenetic Manipulation Coupled with Immunocytochemistry:

  • Express optogenetic regulators of polarity pathways (e.g., photoactivatable CDC42)

  • Stimulate specific neuronal compartments

  • Fix and immunostain for PARD6A at defined intervals post-stimulation

  • Assess redistribution of PARD6A in response to pathway activation

For neuronal studies, consider these specialized protocol modifications:

• Use 4% PFA with 4% sucrose for fixation to better preserve fine neuronal structures

• Extend permeabilization time to 30 minutes with 0.1% Triton X-100 for better antibody penetration

• Implement extended washing steps (6 x 10 minutes) to reduce background in complex neuronal cultures

• Consider using tyramide signal amplification for detecting low-abundance PARD6A in distal neuronal compartments .

How can novel imaging technologies enhance PARD6A localization and interaction studies?

Advanced imaging technologies are revolutionizing our understanding of PARD6A dynamics and interactions:

• Super-Resolution Microscopy Applications:

  • STED (Stimulated Emission Depletion) microscopy: Provides 30-80nm resolution of PARD6A within tight junctions

  • STORM/PALM: Enables single-molecule localization mapping of PARD6A relative to junctional proteins

  • SIM (Structured Illumination Microscopy): Offers improved resolution (100nm) with standard fluorophores

  • Implementation strategy: Use biotin-conjugated primary antibody with bright, photostable fluorophore-conjugated streptavidin suitable for prolonged imaging

• Live-Cell Proximity Labeling:

  • TurboID or miniTurbo fusion with PARD6A expressed in cells

  • Brief biotin pulse labels proteins in proximity to PARD6A

  • Fixed samples can be stained with fluorescent streptavidin alongside PARD6A antibodies

  • Allows temporal mapping of PARD6A interaction networks during polarization events

• Correlative Light and Electron Microscopy (CLEM):

  • Immunofluorescence with biotin-conjugated PARD6A antibodies and fluorescent streptavidin

  • Convert to electron-dense signal using DAB photooxidation

  • Enables ultrastructural localization of PARD6A at membrane domains

• Lattice Light-Sheet Microscopy:

  • Reduced phototoxicity allows extended imaging of PARD6A dynamics

  • Compatible with the "fix and stain" approach at defined timepoints

  • Particularly valuable for 3D cultured systems like organoids

These technologies require specific sample preparation considerations. For super-resolution approaches, researchers should use thinner sections (70-100nm for STORM/PALM), high-quality cover glass (#1.5H), and mounting media matched to the imaging system's refractive index. Additionally, multiplexed imaging may require sequential detection or spectral unmixing to resolve closely positioned signals .

What considerations are important when designing PARD6A knockdown/knockout validation experiments?

Proper validation of PARD6A antibody specificity through knockdown/knockout approaches is critical for research integrity:

• CRISPR/Cas9 Knockout Validation Strategy:

  • Design multiple guide RNAs targeting early exons of PARD6A

  • Generate clonal knockout cell lines and confirm gene disruption by sequencing

  • Perform Western blot and immunofluorescence with PARD6A antibodies

  • Include rescue experiments expressing PARD6A cDNA to confirm phenotype specificity

  • Recommended controls: wild-type parental cells, cells with non-targeting guide RNAs

• RNAi Knockdown Approach:

  • Design siRNA/shRNA targeting different regions of PARD6A mRNA

  • Transfect/transduce cells and confirm knockdown efficiency by qRT-PCR

  • Perform concentration-response experiments to determine optimal knockdown conditions

  • Assess antibody staining intensity quantitatively in knocked-down versus control cells

  • Expected result: proportional reduction in signal intensity corresponding to knockdown level

• Overexpression Controls:

  • Express epitope-tagged PARD6A (e.g., FLAG-PARD6A)

  • Perform dual staining with anti-FLAG and anti-PARD6A antibodies

  • Signals should colocalize in overexpressing cells

Validation data should include:

For PARD6A specifically, researchers should be aware that complete knockout may affect cell viability in some cell types, necessitating the use of inducible knockout systems or careful timing of analyses post-CRISPR editing .

How can researchers integrate PARD6A immunolabeling with high-content screening approaches?

Integrating PARD6A immunolabeling into high-content screening workflows enables large-scale investigation of polarity regulation:

• Automated Immunofluorescence Protocol Development:

  • Optimize biotin-conjugated PARD6A antibody concentration and incubation time for robotic liquid handlers

  • Develop robust washing protocols that maintain sample integrity across 96/384-well formats

  • Standardize detection reagent concentration (streptavidin-fluorophore) for signal consistency

  • Include DAPI and cytoskeletal markers (phalloidin) for automated image segmentation

• Multi-parametric Analysis Approach:

  • Implement machine learning-based image analysis to classify PARD6A distribution patterns

  • Extract quantitative features including:

    • Intensity at cell-cell junctions normalized to cytoplasmic signal

    • Colocalization with polarity markers (aPKC, PAR3)

    • Morphological features of junctional complexes

  • Develop a polarity index incorporating multiple parameters

• Screening Applications:

  • Drug effect profiling on epithelial polarity

  • siRNA/CRISPR screens for novel polarity regulators

  • Patient-derived organoid phenotyping for polarity defects

When implementing high-content approaches, researchers should develop robust quality control metrics to flag technical artifacts, including antibody aggregates, uneven illumination, or segmentation errors. Specialized image analysis pipelines using CellProfiler, FIJI/ImageJ with custom macros, or commercial platforms like Harmony or MetaXpress can be developed for PARD6A-specific applications .

What best practices should researchers follow when working with PARD6A antibodies?

Researchers working with PARD6A antibodies should adhere to rigorous validation and experimental practices to ensure reliable results:

• Comprehensive Antibody Validation:

  • Verify specificity using multiple approaches (Western blot, immunofluorescence, knockout controls)

  • Test across multiple cell types with known PARD6A expression profiles

  • Document lot-to-lot variation for biotin-conjugated antibodies

  • Maintain validation records with images and experimental conditions

• Optimized Experimental Design:

  • Include appropriate positive and negative controls in every experiment

  • For biotin-conjugated antibodies, always include endogenous biotin blocking steps

  • Design experiments with biological replicates across multiple cell lines or primary tissues

  • Standardize image acquisition settings when performing quantitative analyses

• Transparent Reporting:

  • Provide complete methodological details including antibody source, catalog number, lot number, and concentration

  • Document all sample preparation steps, particularly fixation and permeabilization conditions

  • Present unmodified representative images alongside any enhanced or processed images

  • Share detailed analysis workflows including segmentation strategies and quantification parameters

• Consideration of PARD6A Biology:

  • Account for cell-type specific expression patterns and subcellular localization

  • Recognize that PARD6A function depends on its interactions with multiple partners

  • Consider dynamic redistribution during cell division, polarization, and response to stimuli

  • Integrate findings with existing knowledge of polarity complex regulation

By following these best practices, researchers can generate robust and reproducible data that advances our understanding of PARD6A's role in establishing and maintaining cell polarity across diverse physiological and pathological contexts .

What are the most promising future directions for PARD6A antibody applications in biomedical research?

The field of PARD6A research continues to evolve, with several promising directions for antibody applications:

• Single-Cell Analysis of Polarity Heterogeneity:

  • Application of PARD6A antibodies in single-cell proteomics approaches

  • Integration with spatial transcriptomics to correlate protein localization with gene expression

  • Development of multiplexed imaging approaches to simultaneously visualize all components of polarity complexes

  • These approaches will reveal heterogeneity in polarity establishment within tissues and tumors

• Developmental and Stem Cell Applications:

  • Tracking PARD6A dynamics during embryonic development using tissue clearing techniques and whole-mount immunostaining

  • Investigating the role of PARD6A in stem cell asymmetric division and differentiation

  • Correlating PARD6A distribution with developmental outcomes in organoid models

  • These studies will illuminate how polarity establishment guides morphogenesis

• Therapeutic Development:

  • Using PARD6A antibodies to screen for compounds that modulate polarity complex formation

  • Developing high-throughput assays for polarity restoration in cancer models

  • Creating antibody-based tools to monitor polarity restoration during treatment response

  • These applications could lead to novel therapeutic strategies targeting polarity dysregulation

• Advanced Imaging Approaches:

  • Expansion microscopy with PARD6A immunolabeling to visualize nanoscale organization

  • Integration with emerging volumetric imaging techniques for whole-tissue analysis

  • Development of split-epitope approaches for investigating protein conformational changes

  • These techniques will provide unprecedented resolution of polarity complex dynamics