PARD6A Antibody, FITC conjugated

What is PARD6A and why is it significant in cellular research?

PARD6A (Partitioning-defective 6 homolog alpha) functions as a key regulator of cell polarity and tight junction formation. As a vital component of the PAR (Partitioning-defective) complex, it plays a crucial role in establishing and maintaining cell polarity by regulating the localization of proteins involved in cell-cell junctions. PARD6A's significance extends beyond basic cellular architecture, as its dysregulation has been implicated in various diseases, including cancer and neurological disorders, making it a promising target for therapeutic interventions . The protein's central role in the PAR complex, which includes interactions with aPKC and Cdc42, positions it as a critical mediator of polarization processes during epithelial morphogenesis and other cellular events . Researchers investigating cellular architecture, cancer biology, or developmental processes would benefit significantly from studying this protein using specific antibodies.

How do FITC-conjugated PARD6A antibodies differ from unconjugated versions in experimental applications?

FITC-conjugated PARD6A antibodies offer direct visualization capabilities without requiring secondary antibody incubation, streamlining immunofluorescence protocols significantly. While unconjugated PARD6A antibodies (like those described in search results) require a two-step detection process with separate primary and labeled secondary antibodies, FITC-conjugated versions combine both functions in a single reagent .

The methodological differences affect several experimental parameters:

When designing experiments, researchers should consider these differences against their specific experimental needs, particularly when studying PARD6A's cell junction and membrane localization patterns .

What cellular structures can be visualized using PARD6A antibodies?

PARD6A antibodies enable visualization of multiple cellular structures due to the protein's diverse localization pattern. According to cellular localization data, PARD6A is found in:

• Cell junctions - particularly tight junctions where it regulates barrier formation

• Cell membrane - where it participates in polarity complex formation

• Cell projections - including membrane extensions involved in cellular migration

• Cytoplasm - showing diffuse distribution patterns in many cell types

• Membrane ruffles - dynamic structures associated with cell movement

When using FITC-conjugated PARD6A antibodies, researchers should optimize fixation protocols to preserve these structures. For tight junction visualization, methanol fixation often provides superior results compared to paraformaldehyde. For cytoplasmic and membrane ruffle detection, mild permeabilization with 0.1% Triton X-100 yields optimal results while preserving structural integrity .

What is the expected molecular weight of PARD6A in Western blot applications?

When performing Western blot analysis with PARD6A antibodies, researchers should note a significant discrepancy between theoretical and observed molecular weights. The calculated molecular weight of PARD6A is 37kDa, but the observed molecular weight in SDS-PAGE is approximately 47kDa . This 10kDa difference likely results from post-translational modifications such as phosphorylation or other covalent modifications that affect protein migration.

To ensure accurate identification of PARD6A bands:

• Always include appropriate positive controls (e.g., lysates from SW620, HT-29, Jurkat, U-251MG, Raji cells, or mouse testis tissue)

• Use a molecular weight ladder with clear resolution in the 35-50kDa range

• Consider running reduced and non-reduced samples in parallel if studying structural conformation

• Verify specificity through knockdown/knockout validation studies

This information remains relevant when using FITC-conjugated antibodies for other applications, as it confirms target specificity across experimental platforms.

How can experimental design be optimized for studying PARD6A's role in epithelial-mesenchymal transition (EMT)?

PARD6A has been demonstrated to promote epithelial-mesenchymal transition (EMT) in cancer models, particularly in ovarian cancer through a PARD6A-Integrin β1-ILK-SNAIL1 pathway . When designing experiments to investigate this phenomenon, researchers should consider a comprehensive approach:

• Expression analysis methodology:

  • Establish baseline PARD6A expression in normal versus cancer cell lines

  • Correlate expression with cancer stage (PARD6A shows higher expression in advanced stages III-IV)

  • Analyze expression in relation to differentiation status and metastatic potential

• Functional validation approach:

  • Implement both knockdown and overexpression experimental designs

  • For knockdown: siRNA or shRNA targeting PARD6A with appropriate non-targeting controls

  • For overexpression: transfection with PARD6A expression vectors versus empty vector controls

• EMT marker assessment:

  • Monitor E-cadherin (epithelial marker) and VIMENTIN (mesenchymal marker) as primary readouts

  • Include SNAIL1 expression analysis as a key intermediary in the signaling pathway

  • Evaluate Integrin β1 and ILK protein levels as upstream regulators

• Validation through rescue experiments:

  • After PARD6A knockdown, attempt to rescue the EMT phenotype through SNAIL1 overexpression

  • After PARD6A overexpression, assess whether SNAIL1 knockdown blocks EMT induction

This comprehensive experimental design allows for mechanistic dissection of PARD6A's role in EMT while controlling for potential experimental artifacts .

What controls and validation methods are critical when using PARD6A antibodies in research?

When employing PARD6A antibodies (including FITC-conjugated versions) in research applications, implementing rigorous controls and validation methods is essential for generating reliable data:

• Antibody specificity validation:

  • Western blot analysis to confirm single band at expected molecular weight (47kDa observed vs. 37kDa calculated)

  • Peptide competition assays using the immunizing peptide (amino acids 1-100 of human PARD6A for some antibodies)

  • PARD6A knockdown/knockout validation to demonstrate signal reduction

  • Cross-reactivity assessment against other PAR family members (PARD6B, PARD6G)

• Immunofluorescence-specific controls:

  • Secondary antibody-only controls (for unconjugated antibodies) or isotype controls (for FITC-conjugated antibodies)

  • Positive control tissues/cells with known PARD6A expression (SW620, HT-29, Jurkat, U-251MG, Raji, Mouse testis)

  • Autofluorescence controls, particularly important with FITC due to its spectral properties

  • Co-localization with known interacting partners (aPKC, Cdc42) as functional validation

• Functional validation approaches:

  • Correlation of antibody staining patterns with PARD6A mutant phenotypes

  • Antibody detection of differential expression following experimental manipulation

  • Comparison of staining patterns across multiple antibodies targeting different PARD6A epitopes

Implementation of these validation approaches significantly enhances data reliability and facilitates accurate interpretation of experimental outcomes.

How do mutations in PARD6A domains affect protein function and antibody recognition?

Understanding the relationship between PARD6A domain mutations, protein functionality, and antibody epitope recognition is critical for advanced research applications:

When using antibodies to detect PARD6A variants or mutants in experimental systems:

• Verify whether the antibody's epitope overlaps with mutation sites

• For FITC-conjugated antibodies, test detection efficiency against wildtype and mutant proteins

• Consider using multiple antibodies targeting different epitopes when studying PARD6A mutants

• Implement appropriate positive controls for each mutant construct

This approach ensures accurate interpretation of results when investigating the structure-function relationship of PARD6A domains .

What methodological approaches are optimal for multiplexing FITC-conjugated PARD6A antibodies with other markers?

Multiplexing FITC-conjugated PARD6A antibodies with other markers requires careful consideration of spectral overlap, antibody compatibility, and imaging parameters:

• Fluorophore selection strategy:

  • Pair FITC (excitation ~495nm, emission ~520nm) with spectrally distant fluorophores

  • Optimal companions include far-red fluorophores (Cy5, Alexa Fluor 647) and red fluorophores (Texas Red, Alexa Fluor 594)

  • Avoid Rhodamine derivatives due to potential spectral bleed-through with FITC

• Target selection for co-localization studies:

  • For PARD6A function analysis: Co-stain with aPKC and Cdc42 to visualize the complete polarity complex

  • For EMT studies: Combine with E-cadherin and VIMENTIN markers to assess correlation with transition states

  • For cancer progression analysis: Include proliferation markers (Ki-67) and stage-specific markers

• Optimized imaging protocol:

  • Sequential scanning rather than simultaneous acquisition to minimize cross-talk

  • Narrow bandpass filter sets to reduce spectral overlap

  • Careful exposure settings to avoid FITC photobleaching, which occurs more rapidly than with many other fluorophores

• Signal validation approach:

  • Single-color controls for each channel to establish baseline signals

  • Fluorescence minus one (FMO) controls to detect spillover

  • Antibody titration series to determine optimal concentration for specific signal-to-noise ratio

These methodological considerations ensure reliable multiplexed detection when using FITC-conjugated PARD6A antibodies in complex experimental designs .

How can non-specific background be minimized when using FITC-conjugated PARD6A antibodies?

FITC-conjugated antibodies, including those targeting PARD6A, can sometimes exhibit background issues that require specific optimization strategies:

• Sample preparation refinement:

  • Implement dual blocking with both 5% normal serum and 1% BSA

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

  • Add 0.1-0.3% Triton X-100 to blocking buffer to improve antibody penetration while reducing non-specific membrane binding

  • Consider using specialized blocking reagents containing IgG fragments to block Fc receptors

• Antibody incubation optimization:

  • Dilute antibody in fresh blocking buffer rather than PBS alone

  • Extend primary antibody incubation time while reducing concentration (1:1000-1:2000 range)

  • Perform incubations at 4°C overnight rather than room temperature

  • Increase wash duration and volume (minimum 5x10 minute washes)

• Tissue/cell-specific considerations:

  • For tissues with high autofluorescence, pretreat with sodium borohydride (10mg/ml) before blocking

  • When working with cell lines known to express PARD6A (SW620, HT-29, Jurkat, U-251MG, Raji) , use lower antibody concentrations

  • For tissues with low PARD6A expression, consider signal amplification methods

• Imaging parameter adjustments:

  • Optimize exposure settings on positive control samples first

  • Implement background subtraction based on negative control samples

  • Consider spectral unmixing to separate FITC signal from autofluorescence

These optimization steps significantly improve signal-to-noise ratio when using FITC-conjugated PARD6A antibodies in research applications .

How should experimental design address discrepancies in PARD6A localization patterns?

Researchers sometimes encounter discrepancies in PARD6A localization patterns across different experimental systems or antibodies. A methodical approach to resolving these inconsistencies includes:

• Systematic fixation comparison:

  • Compare multiple fixation methods (4% PFA, methanol, acetone) on the same sample

  • Evaluate how each method preserves specific cellular compartments where PARD6A resides (cell junctions, membrane, cytoplasm, ruffles)

  • Document differences in staining patterns with standardized imaging parameters

• Epitope accessibility assessment:

  • Test different permeabilization conditions (0.1% vs. 0.5% Triton X-100, saponin, digitonin)

  • Evaluate whether certain cellular compartments show differential antibody penetration

  • Consider antigen retrieval methods for formalin-fixed tissues

• Cell state and context evaluation:

  • Analyze PARD6A localization under different cell states (confluent vs. sparse, polarized vs. non-polarized)

  • Document dynamic changes during cell cycle progression

  • Assess localization changes during EMT or other transitional states

• Cross-validation strategy:

  • Compare FITC-conjugated antibody patterns with unconjugated antibodies plus fluorescent secondary detection

  • Utilize antibodies targeting different PARD6A epitopes (N-terminal vs. C-terminal)

  • Correlate immunofluorescence patterns with subcellular fractionation and Western blot analysis

This systematic approach helps distinguish genuine biological variability from technical artifacts when analyzing PARD6A localization patterns .

What strategies can resolve contradictory results in PARD6A functional studies?

When faced with contradictory results in PARD6A functional studies, researchers should implement a structured troubleshooting approach:

• Domain-specific functional analysis:

  • Determine whether contradictions relate to specific protein domains

  • Utilize domain-specific mutants (K19A for PB1 domain, ΔPro136 for CRIB domain, M235W for PDZ domain)

  • Assess whether contradictions arise from differential effects on distinct interaction partners

• Cell-type dependent validation:

  • Systematically compare findings across multiple cell types

  • Evaluate baseline PARD6A expression and localization in each model

  • Consider compensation by other PAR family members in different cellular contexts

• Signaling pathway cross-talk assessment:

  • Investigate whether contradictions relate to context-dependent interactions with other pathways

  • Specifically analyze PARD6A interactions with the SNAIL1 pathway and effects on EMT markers

  • Evaluate how experimental perturbations of Integrin β1-ILK signaling affect outcomes

• Reconciliation through comprehensive experimental design:

  • Implement both loss-of-function and gain-of-function approaches in parallel

  • Conduct time-course analyses to capture dynamic effects that may appear contradictory at single timepoints

  • Analyze dose-dependency of effects, particularly for overexpression studies

This structured approach helps resolve apparent contradictions by identifying context-dependent aspects of PARD6A function and methodological variables that influence experimental outcomes .

How can PARD6A antibodies advance understanding of cancer progression mechanisms?

PARD6A antibodies, including FITC-conjugated versions, offer powerful tools for investigating cancer progression mechanisms through several innovative approaches:

• Clinical correlation studies:

  • Analyze PARD6A expression patterns across cancer stages (I-II vs. III-IV) using tissue microarrays

  • Correlate expression levels with differentiation status and lymphatic metastasis

  • Develop prognostic assays based on PARD6A expression patterns and subcellular localization

• Mechanistic dissection of EMT regulation:

  • Track dynamic changes in PARD6A localization during EMT induction

  • Monitor co-localization with SNAIL1 and other EMT regulators during transition states

  • Evaluate the temporal sequence of events in the PARD6A-Integrin β1-ILK-SNAIL1 pathway

• Therapeutic target validation:

  • Use PARD6A antibodies to assess target engagement in drug development pipelines

  • Implement high-content screening approaches to identify compounds disrupting PARD6A interactions

  • Develop antibody-based imaging for in vivo tracking of PARD6A-expressing cancer cells

• Personalized medicine applications:

  • Stratify patient samples based on PARD6A expression patterns

  • Correlate expression with treatment responses and outcomes

  • Develop companion diagnostic approaches for targeted therapies

These research directions leverage the specificity of PARD6A antibodies to translate fundamental understanding of PARD6A biology into clinical applications for cancer diagnosis and treatment .

What methodological advances could improve PARD6A research using conjugated antibodies?

Several emerging methodological approaches could significantly enhance PARD6A research using conjugated antibodies:

• Advanced imaging technologies:

  • Super-resolution microscopy (STORM, PALM, SIM) to resolve PARD6A localization within tight junctions and polarity complexes

  • Live-cell imaging with photoconvertible or photoactivatable fluorophore conjugates to track PARD6A dynamics

  • Correlative light-electron microscopy to link fluorescence patterns with ultrastructural features

• Multiplexed detection systems:

  • Cyclic immunofluorescence to assess PARD6A in relation to dozens of other markers on the same sample

  • Mass cytometry or imaging mass cytometry using metal-tagged antibodies for highly multiplexed analysis

  • Spatial transcriptomics combined with protein detection to correlate PARD6A protein expression with transcriptional programs

• Proximity-based interaction analysis:

  • Development of split-fluorescent protein complementation assays for detecting PARD6A interactions

  • Proximity ligation assays to visualize PARD6A interactions with aPKC, Cdc42, and other partners

  • FRET-based biosensors to monitor PARD6A conformational changes during signaling events

• Therapeutic development platforms:

  • Antibody-drug conjugates targeting PARD6A in cancer cells with elevated expression

  • Intrabodies derived from research-grade antibodies to disrupt specific PARD6A interactions

  • Proteolysis-targeting chimeras (PROTACs) validated using antibody-based detection systems

These methodological advances would expand the utility of PARD6A antibodies beyond detection toward functional manipulation and therapeutic applications .