ZIC3 Antibody

What is the expected molecular weight of ZIC3 in Western blot analysis?

ZIC3 typically appears as a specific band at approximately 56 kDa when detected via Western blot under reducing conditions . Experimental validation shows consistent detection at this molecular weight across multiple cell types, including 293T human embryonic kidney cells, Jurkat human acute T cell leukemia cells, and NIH-3T3 mouse embryonic fibroblast cell lines . When performing Western blots for ZIC3 detection, it is recommended to:

• Use PVDF membranes for optimal protein retention

• Apply approximately 1 μg/mL of primary ZIC3 antibody

• Follow with appropriate HRP-conjugated secondary antibody (such as Anti-Sheep IgG for sheep-derived primary antibodies)

• Ensure proper reducing conditions and buffer selection (e.g., Immunoblot Buffer Group 1)

What is the subcellular localization of ZIC3 and how can this be verified?

ZIC3 demonstrates predominantly nuclear localization in mammalian cells, consistent with its function as a transcription factor . This localization can be verified through immunocytochemistry or immunofluorescence techniques. In the A172 human glioblastoma cell line, ZIC3 has been shown to specifically localize to nuclei using fluorescent immunocytochemistry . A recommended protocol includes:

• Immersion fixation of cultured cells

• Application of 10 μg/mL ZIC3 antibody for 3 hours at room temperature

• Visualization using fluorophore-conjugated secondary antibodies (e.g., NorthernLights™ 557-conjugated Anti-Sheep IgG)

• Counterstaining nuclei with DAPI for reference

• Confirmation of specific nuclear staining pattern

How can cross-reactivity with other ZIC family proteins be assessed?

Given the high sequence homology within the ZIC family (particularly between ZIC2 and ZIC3), cross-reactivity assessment is crucial for experimental validity . The antibody should be validated in knockout or knockdown systems to ensure specificity. Evidence from the literature suggests potential cross-reactivity issues:

• Anti-ZIC3 antibody may recognize ZIC2 or ZIC5 on chromatin in certain contexts

• ChIP-seq for ZIC3 is recommended to be conducted in ZIC2−/−ZIC5−/− backgrounds to ensure specificity

• When possible, epitope-tagged versions (e.g., Flag-ZIC3) can provide additional specificity controls

To assess cross-reactivity:

• Perform Western blots against recombinant ZIC family proteins

• Test antibody in genetic knockout models for each ZIC family member

• Conduct peptide competition assays with specific peptide sequences unique to each ZIC family member

How can ZIC3 antibodies be used to investigate pluripotency transitions?

ZIC3 antibodies are valuable tools for studying the transition from naive to primed pluripotency in embryonic stem cells. The expression of ZIC3 shows a transient increase during this transition, making it an important marker for developmental progression . Methodological approaches include:

Temporal profiling approach:

• Collect embryonic stem cells at different time points during differentiation (e.g., day 0, day 1, day 2)

• Perform Western blot analysis using ZIC3 antibodies to track protein expression changes

• Correlate protein levels with morphological and transcriptional changes

• Compare with other pluripotency markers to establish temporal relationships

In mouse ESCs transitioning to epiblast-like cells (EpiLCs), ZIC3 protein levels peak at day 1 of differentiation, even more prominently than observed at the mRNA level . This temporal pattern can be used to identify transition states between naive and primed pluripotency.

What considerations are important for ChIP-seq experiments using ZIC3 antibodies?

Chromatin immunoprecipitation sequencing (ChIP-seq) with ZIC3 antibodies has revealed important insights into its genomic binding patterns and regulatory functions . Key considerations include:

Experimental design:

• Cell state selection: ZIC3 binding differs between naive and primed states, with highest activity during transition states

• Fixation conditions: Standard 1% formaldehyde for 10 minutes at room temperature

• Sonication parameters: Optimize to achieve 200-500bp DNA fragments

• Antibody selection: Use ChIP-validated antibodies with demonstrated specificity

• Controls: Include IgG control and, if possible, ZIC3 knockout/knockdown controls

Data analysis considerations:

• High confidence ZIC3 binding regions typically number ~4,000-5,000 in transition-state cells

• Majority of binding sites locate in inter- and intra-genic regions

• Motif enrichment analysis should identify the ZIC binding motif alongside other factors like ESSRB and SOX proteins

• Target gene assignment requires careful consideration (nearest-neighbor model has been used successfully)

How can ZIC3 antibodies help investigate interactions with chromatin remodeling complexes?

Recent research highlights ZIC3's role in recruiting the SWI/SNF complex to maintain chromatin accessibility at specific enhancers . Methodological approaches include:

Co-immunoprecipitation (Co-IP):

• Prepare nuclear extracts from relevant cell types (e.g., primed hESCs)

• Immunoprecipitate with ZIC3 antibodies under mild conditions to preserve protein-protein interactions

• Probe for SWI/SNF components (particularly BRG1) in the immunoprecipitate

• Perform reciprocal IP with BRG1 antibodies and detect ZIC3

Sequential ChIP (ChIP-reChIP):

• Perform first ChIP with ZIC3 antibody

• Elute complexes under mild conditions

• Perform second ChIP on the eluate using antibodies against SWI/SNF components

• Sequence or qPCR analyze the resulting DNA to identify co-occupied regions

ATAC-seq in ZIC3-depleted cells:
Accessibility studies show that ZIC3 depletion (especially combined with ZIC2 depletion) leads to decreased accessibility at primed-specific enhancers , supporting a role in maintaining open chromatin via SWI/SNF recruitment.

What approaches can be used to study the functional redundancy between ZIC2 and ZIC3?

The research indicates significant functional overlap between ZIC2 and ZIC3, with dual knockout causing more severe phenotypes than single knockouts . Methodological approaches to study this redundancy include:

Sequential genetic manipulation:

• Generate single knockouts of ZIC2 or ZIC3

• Perform acute ablation of the remaining factor using Cas9 RNP electroporation

• Analyze phenotypes at defined time points (e.g., 5 days post-electroporation)

• Compare transcriptional changes across single and double knockout conditions

Rescue experiments:

• In ZIC2/ZIC3 depleted cells, reintroduce either ZIC2 or ZIC3 individually

• Assess the degree of phenotypic rescue

• Identify processes requiring both factors versus those rescued by either factor alone

Domain swap approaches:

• Generate chimeric constructs swapping domains between ZIC2 and ZIC3

• Express these in knockout backgrounds

• Determine which domains confer functional specificity versus redundancy

Research shows that while single ZIC2 or ZIC3 mutants show minimal transcriptome changes, double mutants exhibit dramatic changes including upregulation of PRC2 targets and spontaneous differentiation .

How can ZIC3 antibodies be used to study its role in enhancer regulation?

ZIC3 has been implicated in maintaining accessibility at primed-specific enhancers . Methodological approaches include:

Integrated genomic analysis:

• Perform ChIP-seq for ZIC3 binding sites

• Overlay with ATAC-seq data to identify accessible regions

• Correlate with histone modification data (H3K27ac, H3K4me1) to identify active enhancers

• Integrate with transcriptome data to link enhancer activity to gene expression

Enhancer reporter assays:

• Clone ZIC3-bound putative enhancers upstream of minimal promoter-reporter constructs

• Test enhancer activity in wild-type versus ZIC3-depleted backgrounds

• Perform site-directed mutagenesis of ZIC binding motifs to confirm direct regulation

Research demonstrates that ZIC3-bound enhancers are enriched for developmental processes and signaling pathways, including BMP and STAT pathways .

What methods can address the relationship between ZIC3 and polycomb repressive complex activity?

Evidence suggests interplay between ZIC factors and polycomb repressive complexes, particularly PRC2 . Methodological approaches include:

ChIP-seq comparative analysis:

• Perform ChIP-seq for ZIC3 and PRC2 components (EZH2, SUZ12)

• Analyze genomic overlap and proximity patterns

• Examine H3K27me3 distribution in wild-type versus ZIC3-depleted cells

• Focus on regions showing high H3K27me3 density

Sequential perturbation:

• Inhibit EZH2 (PRC2 catalytic subunit) using small molecules

• Perform ZIC3 ChIP-seq before and after inhibition

• Identify regions where ZIC3 binding is affected by PRC2 activity

Research shows that concomitant deletion of ZIC2 and ZIC3 leads to:

• Upregulation of canonical Polycomb targets

• Reduced EZH2 occupancy at bivalent promoters

• Decreased H3K27me3 at regions normally showing high H3K27me3 density

What are the common issues in ZIC3 detection and how can they be addressed?

Several challenges can arise when working with ZIC3 antibodies:

Low signal intensity:

• Increase antibody concentration (up to 10 μg/mL has been successfully used for ICC)

• Extend incubation time (3+ hours at room temperature or overnight at 4°C)

• Optimize sample preparation to enhance epitope accessibility

• Use signal amplification methods (e.g., tyramide signal amplification)

Cross-reactivity with other ZIC family members:

• Use antibodies raised against less conserved regions (C-terminal regions show greater divergence)

• Validate in known negative control samples or knockout models

• Consider using epitope-tagged versions in overexpression studies

• Perform peptide competition assays to confirm specificity

Nuclear localization challenges:

• Ensure proper permeabilization of nuclear membranes

• Use appropriate fixation methods that preserve nuclear architecture

• Consider detergent optimization for nuclear protein extraction

How should experimental conditions be optimized for different cell types?

Cell type-specific considerations for ZIC3 detection include:

Stem cells vs. differentiated cells:

• Stem cells may require gentler lysis conditions

• Nuclear extraction protocols may need optimization based on nuclear:cytoplasmic ratio

• Expression levels vary dramatically by developmental stage

Cell-type specific expression patterns:

• ZIC3 expression is highly developmental context-dependent

• Expression peaks during naive-to-primed transition in ESCs

• Optimization of detection methods should consider expected expression levels

Fixation considerations:

• Embryonic stem cells: 4% PFA for 15 minutes at room temperature

• Cell lines: 4% PFA or methanol fixation depending on epitope accessibility

• Test multiple fixation protocols when establishing new cell systems

How should ZIC3 ChIP-seq data be interpreted in the context of pluripotency regulation?

Interpreting ZIC3 ChIP-seq data requires careful analysis of binding patterns and correlation with functional outcomes:

Target identification approach:

• Identify high-confidence binding sites (typical threshold: peak score >10)

• Associate peaks with genes using distance-based approaches

• Perform Gene Ontology analysis on target genes

• Cross-reference with expression data from ZIC3 perturbation experiments

Key findings from ZIC3 ChIP-seq data interpretation:

• ZIC3 binding sites are associated with genes enriched in GO terms for differentiation processes and signaling pathways (BMP, STAT)

• ZIC binding motif is highly enriched in bound regions

• Co-enrichment with motifs for pluripotency factors (ESSRB, SOX) suggests cooperative regulation

• Target genes often encode other transcription factors, indicating a regulatory network position

How can ZIC3 antibodies help distinguish between its activator and repressor functions?

ZIC3 can function as both an activator and repressor depending on genomic context . Methodological approaches to distinguish these roles:

Integrated genomic analysis:

• Combine ZIC3 ChIP-seq with RNA-seq following ZIC3 depletion

• Classify targets as activated (decreased expression upon ZIC3 knockdown) or repressed (increased expression)

• Analyze chromatin features at activation versus repression sites

• Identify potential co-factors specific to each functional mode

Existing data suggests:

• Approximately 53% of ZIC3-regulated genes show reduced expression following ZIC3 depletion, consistent with an activator role

• ZIC3-activated genes show increased expression during progression toward primed state

• This activation function appears dependent on ZIC3 expression, as cells not co-expressing ZIC3 fail to upregulate these targets