PITX3 Recombinant Monoclonal Antibody

What is PITX3 and why is it significant in research?

PITX3 is a homeodomain-containing transcription factor belonging to the RIEG/PITX homeobox family in the bicoid class of homeodomain proteins. It plays critical roles in eye development and differentiation of dopaminergic neurons. PITX3 is essential for normal lens development and differentiation during eye formation in vertebrates . Additionally, it functions as a key transcriptional regulator for the differentiation and maintenance of meso-diencephalic dopaminergic (mdDA) neurons during development and continues to support their long-term survival and maintenance . PITX3 has gained significant research interest due to its associations with congenital eye disorders and potential implications in neurodegenerative conditions affecting dopaminergic neurons.

What are the key applications for PITX3 recombinant monoclonal antibodies?

PITX3 recombinant monoclonal antibodies are primarily utilized in several key applications:

• Western Blot (WB): For detection of PITX3 protein in cell and tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of PITX3

• Flow Cytometry: For detection of intracellular PITX3 in cell populations

• Immunocytochemistry/Immunofluorescence (ICC/IF): For visualization of cellular localization of PITX3

These applications enable researchers to investigate PITX3 expression, localization, and function in various experimental settings, contributing to our understanding of its role in development and disease.

What species reactivity can I expect with commercially available PITX3 antibodies?

Most commercially available PITX3 recombinant monoclonal antibodies demonstrate confirmed reactivity with human PITX3 . Some antibodies also cross-react with mouse and rat PITX3 due to high sequence homology across these species . When selecting an antibody for your research, it's important to verify the species reactivity in the product documentation. For example, the PITX3 rabbit monoclonal antibody (A19261) has confirmed cross-reactivity with human, mouse, and rat samples , while other antibodies may have more limited species reactivity profiles.

How should I optimize immunocytochemistry protocols for PITX3 detection in different cell types?

For optimal immunocytochemistry/immunofluorescence detection of PITX3, consider the following methodological approach:

• Fixation: Fix cells in 4% formaldehyde (paraformaldehyde) for 10-15 minutes at room temperature to preserve cellular architecture while maintaining antigen accessibility .

• Permeabilization: Permeabilize the fixed cells with 0.2% Triton X-100 for 10 minutes to allow antibody access to nuclear PITX3 .

• Blocking: Block non-specific binding sites with 10% normal goat serum (or appropriate serum based on secondary antibody species) for 30-60 minutes .

• Primary antibody incubation: Dilute PITX3 antibody to the recommended concentration (typically 1:50-1:200 for immunocytochemistry applications) and incubate overnight at 4°C for optimal binding .

• Secondary antibody: Use fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 520-conjugated Goat Anti-Rabbit IgG) at 1:200-1:500 dilution for 35-60 minutes at room temperature .

• Counterstaining: Include DAPI nuclear counterstain to facilitate visualization of nuclear localization of PITX3 .

• Controls: Always include appropriate negative controls (omitting primary antibody or using isotype control) and positive controls (cell lines known to express PITX3) to validate specificity .

For neuronal cells or lens epithelial cells specifically, pre-treatment with protease inhibitors may improve signal quality due to the high proteolytic activity in these cell types.

What are the critical parameters for Western blot analysis of PITX3?

For successful Western blot detection of PITX3, consider these critical parameters:

• Sample preparation: PITX3 is predominantly nuclear; therefore, ensure efficient nuclear protein extraction. Use appropriate lysis buffers with protease inhibitors to prevent degradation .

• Loading amount: Load 25-30 μg of total protein per lane for cell lysates to ensure adequate PITX3 detection .

• Expected molecular weight: Look for PITX3 bands at approximately 32-37 kDa. The calculated molecular weight is 32 kDa, but the observed molecular weight is typically around 35-37 kDa due to post-translational modifications .

• Antibody dilution: Use optimal dilution ratios for PITX3 primary antibodies (generally 1:500-1:1000 for Western blot applications) .

• Blocking agent: 3-5% non-fat dry milk or BSA in TBST is typically effective for reducing background signal .

• Detection system: ECL-based detection systems provide suitable sensitivity for PITX3 detection .

• Exposure time: Optimal exposure times range from 90 seconds to several minutes depending on expression levels and antibody sensitivity .

Remember to include positive controls (cells with known PITX3 expression) and molecular weight markers to validate specificity of your detected bands.

How do I optimize flow cytometry protocols for intracellular detection of PITX3?

For successful flow cytometric analysis of PITX3, implement this methodological approach:

• Cell preparation: Harvest cells in growth phase and wash with PBS to remove media components that could interfere with antibody binding.

• Fixation: Fix cells in 4% formaldehyde for 10-15 minutes at room temperature to preserve cellular architecture .

• Permeabilization: Since PITX3 is predominantly nuclear, thorough permeabilization with 0.2% Triton X-100 is crucial for antibody access to nuclear targets .

• Blocking: Block with 10% normal goat serum (or appropriate species based on secondary antibody) to reduce non-specific binding .

• Antibody concentration: Titrate the PITX3 antibody; recommended starting dilutions are 1:50-1:200 (approximately 1μg/1×10^6 cells) with incubation for 45 minutes at 4°C .

• Secondary antibody: Use fluorophore-conjugated secondary antibodies (e.g., FITC-conjugated anti-rabbit IgG) at 1:200 dilution with 35-minute incubation at 4°C .

• Controls: Include isotype control antibodies (e.g., rabbit IgG at the same concentration as the primary antibody) to establish appropriate gating strategies .

• Acquisition parameters: Acquire at least 10,000 events for statistically significant results .

• Analysis: Use appropriate gating strategies first for intact cells (FSC vs SSC), then for single cells (doublet discrimination), and finally analyze PITX3 expression in your population of interest.

This approach has been successfully employed for detection of PITX3 in cell lines such as SH-SY5Y neuroblastoma cells .

How can I investigate the functional consequences of PITX3 mutations using antibody-based techniques?

To investigate functional consequences of PITX3 mutations using antibody-based techniques, implement this multi-faceted approach:

• Expression system setup: Generate expression constructs containing wild-type and mutant PITX3 sequences (such as S13N and G219fs mutations) with epitope tags (e.g., myc) to facilitate detection .

• Subcellular localization analysis: Perform immunocytochemistry using anti-PITX3 or anti-epitope tag antibodies to determine if mutations alter nuclear localization patterns. Conduct quantitative analysis by counting cells with exclusive nuclear localization versus those with cytoplasmic distribution .

• DNA-binding capacity assessment: Employ chromatin immunoprecipitation (ChIP) with PITX3 antibodies to compare binding of wild-type versus mutant PITX3 to target gene promoters (e.g., bicoid elements).

• Protein-protein interaction studies: Use co-immunoprecipitation with PITX3 antibodies to investigate if mutations affect interactions with transcriptional cofactors like NR4A2/NURR1 or corepressors such as NCOR2/SMRT .

• Transcriptional activity evaluation: Combine PITX3 antibodies with reporter gene assays to compare transactivation capabilities of wild-type and mutant proteins on target promoters .

• Dominant-negative effect assessment: Co-express wild-type and mutant PITX3 at varying ratios, then use immunoprecipitation and functional assays to determine if mutants exert dominant-negative effects .

This approach has revealed that mutations like S13N and G219fs exhibit altered DNA-binding profiles and/or reduced transactivation activities while still maintaining nuclear localization, representing partial loss-of-function mutations with variable effects in different ocular cell types .

What are the best practices for dual immunostaining to investigate PITX3 co-localization with other transcription factors?

For effective dual immunostaining to investigate PITX3 co-localization with other transcription factors, implement these best practices:

• Primary antibody selection: Choose PITX3 antibodies from different host species than antibodies against your other target transcription factors (e.g., rabbit anti-PITX3 and mouse anti-NR4A2/NURR1) to avoid cross-reactivity.

• Sequential staining protocol:

  • Fix cells in 4% paraformaldehyde for 15 minutes

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

  • Block with 10% serum from both secondary antibody host species

  • Apply first primary antibody (e.g., anti-PITX3 at 1:50-1:100) overnight at 4°C

  • Add corresponding secondary antibody (e.g., Alexa Fluor 488-conjugated)

  • Apply second primary antibody (e.g., anti-NR4A2/NURR1) overnight at 4°C

  • Add corresponding secondary antibody with a different fluorophore (e.g., Alexa Fluor 594-conjugated)

  • Counterstain with DAPI and mount

• Controls: Include single-stained samples for each antibody to verify absence of spectral overlap and bleed-through between channels.

• Image acquisition: Use confocal microscopy with sequential scanning to minimize fluorophore cross-excitation and ensure accurate co-localization assessment.

• Quantitative analysis: Employ co-localization coefficients (e.g., Pearson's or Manders' coefficients) to quantify the degree of spatial overlap between PITX3 and your other transcription factor of interest.

This approach is particularly valuable for studying interactions between PITX3 and factors involved in dopaminergic neuron development (like NR4A2/NURR1) or lens development regulatory networks.

How can PITX3 antibodies be applied in chromatin immunoprecipitation studies?

For successful application of PITX3 antibodies in chromatin immunoprecipitation (ChIP) studies, follow this methodological approach:

• Crosslinking: Crosslink protein-DNA complexes using 1% formaldehyde for 10 minutes at room temperature, followed by quenching with 125 mM glycine.

• Chromatin preparation: Lyse cells, isolate nuclei, and shear chromatin to 200-500 bp fragments using sonication or enzymatic digestion.

• Antibody selection: Choose ChIP-validated PITX3 recombinant monoclonal antibodies that recognize the native conformation of PITX3. Antibodies recognizing epitopes outside the DNA-binding domain are often more successful.

• Pre-clearing: Pre-clear chromatin with protein A/G beads to reduce non-specific binding.

• Immunoprecipitation: Incubate pre-cleared chromatin with 2-5 μg of PITX3 antibody overnight at 4°C, then add protein A/G beads to capture antibody-chromatin complexes.

• Washing and elution: Perform stringent washing steps to remove non-specific binding, then elute chromatin from beads.

• Reverse crosslinking: Reverse formaldehyde crosslinks by heating at 65°C overnight.

• DNA purification: Purify the immunoprecipitated DNA for downstream analysis.

• Analysis methods:

  • ChIP-qPCR: For validation of PITX3 binding to specific target promoters (e.g., SLC6A3, SLC18A2, TH, DRD2, FOXE3)

  • ChIP-seq: For genome-wide identification of PITX3 binding sites

• Controls: Include input chromatin (non-immunoprecipitated), IgG control (non-specific antibody), and positive control (antibody against a known abundant transcription factor) samples.

This approach enables identification of direct PITX3 transcriptional targets and characterization of its regulatory networks in dopaminergic neuron development and lens formation.

How do I resolve weak or absent signal when using PITX3 antibodies in immunodetection methods?

When encountering weak or absent PITX3 signal in immunodetection applications, systematically address these potential issues:

• Antibody-related factors:

  • Antibody concentration: Titrate antibody concentration; try higher concentrations (1:20-1:50) for immunocytochemistry or flow cytometry applications .

  • Incubation conditions: Extend primary antibody incubation to overnight at 4°C to enhance binding .

  • Antibody quality: Check antibody viability and storage conditions; avoid repeated freeze-thaw cycles.

• Sample-related factors:

  • Expression levels: Verify PITX3 expression in your sample type; use positive controls like SH-SY5Y cells or A549 cells known to express PITX3 .

  • Protein degradation: Include protease inhibitors in all steps of sample preparation.

  • Epitope accessibility: Try different antigen retrieval methods for fixed tissues or different fixation protocols.

• Protocol optimization:

  • Permeabilization: Enhance nuclear permeabilization with 0.5% Triton X-100 to improve antibody access to nuclear PITX3 .

  • Blocking optimization: Test alternative blocking agents (BSA vs. serum) to reduce background and improve signal-to-noise ratio.

  • Detection sensitivity: Use signal amplification systems like tyramide signal amplification (TSA) for low abundance targets.

• Application-specific considerations:

  • Western blot: Increase protein loading to 40-50 μg per lane and optimize transfer conditions for nuclear proteins.

  • Immunocytochemistry: Use confocal microscopy with Z-stack acquisition to improve detection of nuclear signals.

  • Flow cytometry: Enhance permeabilization and use brighter fluorophores for detection.

If signal remains problematic, consider using alternative antibody clones targeting different epitopes within PITX3.

How can I differentiate specific from non-specific staining when using PITX3 antibodies?

To differentiate specific from non-specific staining when using PITX3 antibodies, implement these validation strategies:

• Inclusion of appropriate controls:

  • Negative controls: Include isotype control antibodies (e.g., rabbit IgG at equivalent concentration) to establish baseline non-specific binding .

  • Blocking peptide controls: Pre-incubate PITX3 antibody with immunizing peptide (if available) to demonstrate signal specificity.

  • Genetic controls: When possible, use PITX3 knockout or knockdown samples as definitive negative controls.

• Localization pattern analysis:

  • PITX3 is predominantly nuclear; 91-99% of specific staining should be exclusively nuclear .

  • Non-specific staining often presents as diffuse cytoplasmic or membrane patterns.

• Signal validation across multiple techniques:

  • Confirm PITX3 detection using orthogonal methods (e.g., if detected by immunofluorescence, validate with Western blot).

  • Expected molecular weight on Western blot is approximately 32-37 kDa; bands at significantly different sizes may represent non-specific binding .

• Concentration-dependent signal analysis:

  • Perform antibody titration experiments; specific staining should show a dose-dependent relationship with antibody concentration.

  • Non-specific background often does not decrease proportionally with antibody dilution.

• Cell type specificity checks:

  • Compare staining in cell types known to express PITX3 (e.g., lens epithelial cells, dopaminergic neurons) versus those not expressing PITX3.

  • Tissue-specific expression patterns should match known PITX3 biology.

This comprehensive validation approach ensures confident interpretation of PITX3 antibody staining patterns.

What factors should I consider when interpreting variable PITX3 antibody results across different cell lines?

When interpreting variable PITX3 antibody results across different cell lines, consider these critical factors:

• Differential expression levels: PITX3 expression varies naturally between cell types, with higher expression expected in lens epithelial cells, dopaminergic neuronal cells, and certain cancer cell lines. Compare your results with published expression databases to confirm expected patterns.

• Cell type-specific post-translational modifications: PITX3 may undergo different post-translational modifications depending on cell type, potentially affecting epitope accessibility and antibody recognition. This can manifest as:

  • Shifted molecular weight bands in Western blots

  • Variable staining intensity in immunocytochemistry

  • Differential detection in flow cytometry

• Endogenous regulatory factors: Different cell types contain variable levels of PITX3 cofactors that can affect its conformation and antibody accessibility. Research has demonstrated that mutant PITX3 activity varies significantly between lens epithelial and corneal stromal cells, suggesting the presence of cell type-specific cofactors .

• Nuclear transport efficiency: The efficiency of PITX3 nuclear localization may vary between cell types due to differences in nuclear transport machinery, affecting detection of this predominantly nuclear protein .

• Protocol optimization requirements: Different cell types may require specific fixation and permeabilization protocols. For example:

  • Lens epithelial cells: Standard 4% formaldehyde fixation works well

  • Neuronal cells: May require shorter fixation to preserve antigenicity

  • Fibroblasts: May need enhanced permeabilization for nuclear antigen access

• Antibody clone specificity: Different antibody clones recognize distinct epitopes that may be differentially accessible across cell types. Consider testing multiple antibody clones if consistent detection across cell types is required.

This contextual analysis helps distinguish biological variation from technical artifacts when comparing PITX3 antibody results across different cellular systems.

How can PITX3 antibodies be applied to study dopaminergic neuronal development?

PITX3 antibodies can be powerful tools for studying dopaminergic neuronal development through these methodological approaches:

• Developmental timeline analysis: Use PITX3 antibodies to track expression patterns during neuronal differentiation:

  • Perform immunostaining on tissue sections or differentiating stem cells at various developmental timepoints

  • Combine with markers for neuronal progenitors and mature dopaminergic neurons to establish temporal relationships

  • Correlate PITX3 expression with functional maturation of dopaminergic systems

• Lineage tracing studies: Combine PITX3 immunostaining with other dopaminergic markers:

  • Early markers: FOXA2, LMX1A, LMX1B

  • Terminal differentiation markers: TH (tyrosine hydroxylase), DAT (dopamine transporter), VMAT2

  • Create quantitative co-expression matrices to define developmental trajectories

• Mechanistic investigation of PITX3 function:

  • Use PITX3 antibodies for ChIP studies to identify direct transcriptional targets in dopaminergic neuronal precursors

  • Combine with co-immunoprecipitation to identify interaction with NR4A2/NURR1 and reveal how PITX3 decreases NR4A2/NURR1 interaction with the corepressor NCOR2/SMRT

  • Apply in cellular models with PITX3 mutations to study impact on dopaminergic differentiation

• Stem cell differentiation quality control:

  • Use flow cytometry with PITX3 antibodies to quantify the percentage of cells successfully differentiating toward dopaminergic fate

  • Establish PITX3 expression as a quality metric for dopaminergic neurons derived from stem cells for disease modeling or transplantation studies

This integrative approach enables elucidation of PITX3's role in specification, differentiation, and maintenance of dopaminergic neurons, with implications for understanding neurodevelopmental disorders and Parkinson's disease.

What approaches can be used to study PITX3 mutations in lens development disorders?

To study PITX3 mutations in lens development disorders, implement these comprehensive research approaches using antibodies:

• Comparative immunohistochemistry: Analyze expression patterns of wild-type and mutant PITX3 in lens tissue:

  • Compare nuclear localization efficiency between wild-type and mutant PITX3 proteins (S13N, G219fs)

  • Quantify expression levels in different regions of developing lens

  • Correlate expression patterns with morphological abnormalities

• Functional genomics approach:

  • Perform ChIP-seq to identify differential binding sites between wild-type and mutant PITX3

  • Integrate with RNA-seq to correlate binding alterations with gene expression changes

  • Use PITX3 antibodies to validate direct regulation of key targets like FOXE3 (positively regulated) and PROX1 (negatively regulated)

• Cell cycle regulation analysis:

  • Investigate how PITX3 mutations affect lens epithelial cell proliferation

  • Use co-immunostaining with PITX3 antibodies and cell cycle markers (Ki67, PCNA)

  • Correlate with expression of CDKN1B/P27Kip1 and CDKN1C/P57Kip2, which are normally suppressed by PITX3 activity

• Transcriptional activity assessment:

  • Compare transactivation capabilities of wild-type and mutant PITX3 in lens epithelial cells

  • Quantify differences between mutations (e.g., S13N retains ~77% activity while G219fs retains ~46% in lens epithelial cells)

  • Investigate cell type-specific effects (same mutations show different activity profiles in corneal stromal cells)

• Protein-protein interaction studies:

  • Use co-immunoprecipitation with PITX3 antibodies to identify differential protein interactions between wild-type and mutant proteins

  • Focus on interactions affecting crystallin gene regulation, which are crucial for lens transparency

This multimodal approach provides mechanistic insights into how PITX3 mutations lead to congenital cataracts, anterior segment dysgenesis, and other ocular developmental disorders.

How can multiplexed antibody approaches be used to study PITX3 in complex developmental networks?

Multiplexed antibody approaches provide powerful tools for studying PITX3 within complex developmental networks through these methodological strategies:

• Multicolor immunofluorescence mapping:

  • Combine PITX3 antibodies with antibodies against upstream regulators and downstream targets

  • Implement 4-5 color imaging systems using spectrally distinct fluorophores

  • Apply spectral unmixing algorithms for accurate signal separation

  • Create spatial relationship maps of transcription factor networks in developing tissues

• Sequential multiplexed immunohistochemistry:

  • Utilize cyclic immunofluorescence methods with antibody stripping or quenching between rounds

  • Include PITX3 in antibody panels with markers for:

    • Lens development: PAX6, SOX2, FOXE3, PROX1, crystallins

    • Dopaminergic development: LMX1A, EN1, NR4A2/NURR1, TH, DAT

  • Generate comprehensive tissue maps with 10-20 markers on the same section

• Mass cytometry (CyTOF) analysis:

  • Label PITX3 antibodies with rare earth metals

  • Combine with other metal-labeled antibodies to simultaneously detect 30-40 proteins

  • Perform high-dimensional analysis of developmental trajectories

  • Apply dimensionality reduction techniques (tSNE, UMAP) to visualize complex relationships

• Spatial transcriptomics integration:

  • Correlate PITX3 protein localization with spatial transcriptomic data

  • Map protein expression to transcriptional territories

  • Identify zones of active PITX3-mediated transcription versus protein presence

• Quantitative interaction proteomics:

  • Use PITX3 antibodies for immunoprecipitation followed by mass spectrometry

  • Compare interactomes across developmental stages

  • Identify context-specific cofactors explaining tissue-specific PITX3 functions

This integrated approach reveals how PITX3 operates within gene regulatory networks governing lens development and dopaminergic neuron specification, providing systems-level insights into developmental processes and potential therapeutic targets for associated disorders.

Comparison of PITX3 Mutant Functional Activities in Different Cell Types

This comparative data demonstrates the differential effects of PITX3 mutations across cell types and functional parameters, highlighting the context-dependent nature of mutation effects .