ZNF462 Antibody

What is ZNF462 and why is it significant for research?

ZNF462 is a vertebrate-specific C₂H₂-type zinc finger protein that functions in chromatin organization and transcriptional regulation. The human version has a canonical amino acid length of 2506 residues and a protein mass of 284.7 kilodaltons, with three identified isoforms . It is primarily localized in the nucleus of cells where it plays crucial roles in development . The significance of ZNF462 stems from its recently discovered association with Weiss-Kruszka Syndrome (OMIM#: 618619), a neurodevelopmental disorder characterized by craniofacial anomalies and developmental delay .

Research has shown that ZNF462 safeguards neural lineage specification by targeting the G9A/GLP histone methyltransferase complex to silence mesoendodermal genes in mouse embryonic stem cells . This epigenetic regulation function appears critical for proper development, as haploinsufficiency of ZNF462 results in disrupted embryonic development, contributing to the aforementioned clinical phenotypes .

What experimental applications are suitable for ZNF462 antibodies?

ZNF462 antibodies can be utilized in multiple research applications with varying protocol optimizations. The most common applications include:

• Western Blot (WB): Effective for detecting ZNF462 protein expression levels and changes under various experimental conditions .

• Enzyme-Linked Immunosorbent Assay (ELISA): Useful for quantitative measurement of ZNF462 in solution .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Valuable for examining subcellular localization and co-localization with other proteins .

• Immunohistochemistry-Paraffin (IHC-P): Important for tissue distribution studies and developmental expression analysis .

When selecting antibodies for these applications, researchers should consider the species reactivity required for their model system. Available antibodies show reactivity to human, mouse, and rat ZNF462 with varying degrees of cross-reactivity .

How should ZNF462 antibodies be stored and handled?

For optimal preservation of antibody function, ZNF462 antibodies should be stored at 4°C in the dark and should not be frozen . Most commercial ZNF462 antibodies are supplied in PBS formulation without preservatives, requiring careful handling to prevent contamination .

The stability of these antibodies is typically guaranteed for one year from the date of receipt when stored properly . Before each use, antibodies should be gently mixed by inverting the vial several times rather than vortexing, which can damage the antibody structure. Optimal dilutions for specific applications should be determined experimentally, as recommended concentrations can vary between manufacturers and applications .

What controls should be included when working with ZNF462 antibodies?

Implementing proper controls is essential for generating reliable data when working with ZNF462 antibodies:

• Positive Controls: Include tissue or cell lines known to express ZNF462, such as neural progenitor cells where ZNF462 expression is highest .

• Negative Controls:

  • Primary antibody omission

  • Isotype controls matching the ZNF462 antibody class

  • Tissues from ZNF462 knockout models when available

• Validation Controls:

  • Peptide competition assays using the immunogen sequence (HGAALNTEKRFPCEFCGRAFSQGSEWERHVLRHGMALNDTKQVSREEIHPKEIMENSVKMPSIEEKEDDEAIGIDFSLKNETVAICVVTA)

  • RNA interference experiments to confirm signal specificity

When reporting results, include images of both experimental and control conditions to demonstrate antibody specificity.

How can researchers optimize Western blot protocols for ZNF462 detection?

Detecting ZNF462 by Western blot requires specific optimization due to its high molecular weight (284.7 kDa) :

Recommended Protocol Modifications:

• Sample Preparation:

  • Use phosphatase inhibitors in addition to protease inhibitors in lysis buffers

  • Avoid repeated freeze-thaw cycles of protein samples

  • Heat samples at 70°C for 10 minutes instead of 95°C to prevent aggregation of large proteins

• Gel Electrophoresis:

  • Utilize gradient gels (4-12%) to better resolve high molecular weight proteins

  • Extend running time at lower voltage (80-100V) to improve separation

• Transfer Parameters:

  • Implement wet transfer systems rather than semi-dry for large proteins

  • Use reduced methanol concentration (10% instead of 20%) in transfer buffer

  • Extend transfer time to 16 hours at 30V at 4°C

• Detection:

  • Increase primary antibody incubation time to overnight at 4°C

  • Optimize antibody dilution through titration experiments

  • Use enhanced chemiluminescence systems with extended exposure times

These modifications help overcome the challenges associated with detecting high molecular weight nuclear proteins like ZNF462.

How can ZNF462 antibodies be used to study its role in chromatin regulation?

ZNF462 functions as a critical component in chromatin regulation through its interaction with the G9A/GLP histone methyltransferase complex . To investigate this role, researchers can employ several advanced techniques:

• Chromatin Immunoprecipitation (ChIP):

  • Use ZNF462 antibodies to identify genomic binding sites, particularly at transposable elements that harbor ESC-specific transcription factor binding sites

  • Combine with sequencing (ChIP-seq) to generate genome-wide binding profiles

  • Compare ZNF462 binding profiles with H3K9me2 marks to establish correlation

• Co-Immunoprecipitation (Co-IP):

  • Confirm interactions between ZNF462 and known partners (HP1γ, G9A, GLP, and WIZ)

  • Identify novel interaction partners in different cell types or developmental stages

  • Use reciprocal Co-IP with Avi-tagged proteins as demonstrated with Avi-GLP

• Proximity Ligation Assay (PLA):

  • Visualize ZNF462-G9A/GLP interactions in situ

  • Quantify spatial and temporal changes in these interactions during differentiation

• ATAC-seq Analysis in ZNF462-depleted Cells:

  • Investigate changes in chromatin accessibility at ZNF462 target sites

  • Correlate with gene expression changes, particularly of mesoendodermal genes

These methodologies can provide mechanistic insights into how ZNF462 recruits epigenetic regulators to specific genomic loci to maintain cell identity.

What are the considerations for studying ZNF462 in neurodevelopmental contexts?

Given ZNF462's association with neurodevelopmental disorders and its high expression in neural progenitor cells , researchers investigating its role in neural development should consider:

• Developmental Time-course Analysis:

  • Track ZNF462 expression during neural differentiation, noting that expression levels increase during neural differentiation with highest levels in NPCs

  • Use immunohistochemistry with ZNF462 antibodies on brain sections from different developmental stages

• Disease Modeling:

  • Compare ZNF462 expression and localization in samples from patients with Weiss-Kruszka Syndrome versus controls

  • Use CRISPR-Cas9 to generate heterozygous mutations (R1257* or similar) that mimic human disease variants

• Neural Differentiation Assays:

  • Implement the established protocol transitioning from naïve ground state ESCs to NPCs via embryoid bodies and retinoic acid treatment

  • Compare wild-type, heterozygous, and homozygous ZNF462 mutant cells to assess differentiation capacity

• Single-cell Analysis:

  • Combine ZNF462 immunostaining with markers of neural progenitor subtypes

  • Assess co-expression patterns to identify cell populations most affected by ZNF462 dysfunction

This focused approach can reveal how ZNF462 dysfunction leads to the specific neurodevelopmental phenotypes observed in patients.

How can researchers address epitope masking when detecting ZNF462 in different cellular contexts?

ZNF462 functions within multi-protein complexes that may obscure antibody epitopes in certain cellular contexts. To address this challenge:

• Epitope Retrieval Optimization:

  • Compare different antigen retrieval methods (heat-induced vs. enzymatic) for fixed samples

  • Test multiple buffer systems (citrate, EDTA, Tris) at varying pH levels

  • Adjust retrieval times based on tissue type and fixation duration

• Multiple Antibody Approach:

  • Use antibodies targeting different epitopes within ZNF462

  • Compare staining patterns to identify potential protein interaction domains

  • The immunogen sequence (HGAALNTEKR...VAICVVTA) represents only a small portion of the 2506-residue protein

• Protein Complex Disruption:

  • Apply mild detergents or increased salt concentrations during sample preparation

  • Consider sequential extraction protocols to release ZNF462 from tightly bound chromatin

  • Use protein crosslinking before immunoprecipitation to capture transient interactions

• Signal Amplification Techniques:

  • Implement tyramide signal amplification for low abundance detection

  • Use proximity ligation assays to detect ZNF462 even when some epitopes are masked

  • Consider super-resolution microscopy for detailed localization studies

These approaches can help overcome the challenge of detecting ZNF462 in various cellular contexts where protein interactions may interfere with antibody binding.

How should researchers interpret discrepancies between ZNF462 mRNA and protein expression data?

Researchers may encounter situations where ZNF462 mRNA levels do not correlate with protein detection by antibodies. Several factors may contribute to these discrepancies:

• Post-transcriptional Regulation:

  • ZNF462 may be subject to microRNA regulation or RNA binding protein-mediated control

  • Assess transcript stability through actinomycin D chase experiments

  • Investigate alternative splicing that might affect antibody epitope presence

• Post-translational Modifications:

  • ZNF462 may undergo modifications affecting antibody recognition

  • Phosphorylation, ubiquitination, or SUMOylation could mask epitopes

  • Compare detection using antibodies targeting different regions of the protein

• Protein Stability and Turnover:

  • Determine ZNF462 protein half-life through cycloheximide chase assays

  • Investigate proteasome-dependent degradation by applying MG132 treatment

  • Compare protein levels in different subcellular fractions to assess compartmentalization

• Technical Considerations:

  • Validate RNA measurement techniques (qPCR, RNA-seq) and protein detection methods

  • Ensure samples are prepared from equivalent cell numbers and developmental stages

  • Consider temporal delays between transcription and translation

When facing such discrepancies, a multi-faceted approach combining transcriptomic, proteomic, and cell biological techniques is recommended for accurate interpretation.

What are common pitfalls when interpreting ZNF462 knockout/knockdown phenotypes?

When analyzing phenotypes resulting from ZNF462 manipulation, researchers should consider several potential confounding factors:

• Compensatory Mechanisms:

  • Related zinc finger proteins may partially compensate for ZNF462 loss

  • Acute (knockdown) versus chronic (knockout) loss may exhibit different phenotypes

  • Temporal induction systems can help distinguish direct from compensated effects

• Heterogeneity in Cellular Responses:

  • Single-cell approaches may reveal population heterogeneity masked in bulk analyses

  • Compare complete knockout with heterozygous models that mimic human disease

  • Consider analyzing phenotypes across multiple time points after ZNF462 depletion

• Secondary Effects versus Direct Targets:

  • Combine ZNF462 ChIP-seq with RNA-seq after knockout/knockdown to distinguish direct from indirect effects

  • Acute manipulation (e.g., auxin-inducible degradation) can help identify primary consequences

  • Rescue experiments with full-length versus domain mutants can pinpoint critical functional regions

• Context Dependency:

  • ZNF462 function appears to be cell-type and developmental stage-specific

  • Compare phenotypes across multiple cell types and differentiation states

  • Culture conditions (e.g., 2i/S/L versus standard conditions) affect ZNF462 expression and function

Understanding these considerations can help researchers correctly interpret complex phenotypes associated with ZNF462 dysfunction and distinguish mechanistic insights from technical artifacts.

How might ZNF462 antibodies be used to investigate its role in Weiss-Kruszka Syndrome?

Weiss-Kruszka Syndrome, associated with ZNF462 haploinsufficiency, presents opportunities for translational research using ZNF462 antibodies:

• Patient-derived Cell Models:

  • Compare ZNF462 localization and chromatin association in patient-derived cells versus controls

  • Investigate differential binding partners in affected versus unaffected tissues

  • Correlate protein expression levels with severity of clinical phenotypes

• Developmental Timing Analysis:

  • Use ZNF462 antibodies to track expression during critical developmental windows in model organisms

  • Correlate temporal-spatial expression patterns with the emergence of syndrome-specific features

  • Compare wild-type with heterozygous models mimicking the R1257* or R1263* patient mutations

• Therapeutic Screening Applications:

  • Screen for compounds that might enhance residual ZNF462 function

  • Identify drugs that modulate downstream pathways affected by ZNF462 deficiency

  • Use antibodies to monitor treatment effects on protein levels and localization

• Structure-Function Analysis:

  • Map critical functional domains through comparison of truncated proteins

  • Correlate specific mutations with disruption of particular protein interactions

  • Develop domain-specific antibodies to track functionality of mutation-containing ZNF462

These approaches could provide mechanistic insights and potential therapeutic avenues for patients with Weiss-Kruszka Syndrome.

What emerging technologies might enhance ZNF462 antibody-based research?

Several cutting-edge technologies could significantly advance ZNF462 research:

• Proximity Proteomics:

  • BioID or APEX2 fusions with ZNF462 can identify proximal interactors in living cells

  • Compare interaction networks across developmental stages and in disease models

  • Integrate with existing co-immunoprecipitation data to build comprehensive interaction maps

• Live-Cell Antibody Fragments:

  • Develop cell-permeable nanobodies or single-chain antibody fragments against ZNF462

  • Track protein dynamics in real-time during differentiation or stress responses

  • Combine with optogenetic tools for spatiotemporal control of ZNF462 function

• Spatial Transcriptomics Integration:

  • Correlate ZNF462 protein localization with spatial transcriptomics data

  • Map the impact of ZNF462 on gene expression patterns in developing tissues

  • Combine with single-cell approaches to link protein presence with transcriptional states

• Cryo-EM Structural Analysis:

  • Use antibodies to stabilize ZNF462 complexes for structural determination

  • Generate structural insights into how ZNF462 recruits G9A/GLP to chromatin

  • Design structure-based therapeutic approaches for Weiss-Kruszka Syndrome

These technologies, when combined with existing ZNF462 antibodies, could significantly expand our understanding of this protein's function in health and disease.