ZNF292 Antibody

What is ZNF292 and why is it important to study?

ZNF292 is a zinc finger protein that functions as a transcription factor. It has gained research significance due to two major roles:

• Neurodevelopmental function: De novo and inherited variants in ZNF292 are associated with intellectual disability (ID), autism spectrum disorder (ASD), and other neurodevelopmental features . ZNF292 is highly expressed in the developing human brain, particularly in the cerebellum during the prenatal period, suggesting its critical role in neurodevelopment .

• Tumor suppressor activity: ZNF292 has been identified as a tumor suppressor with roles in cancer development and progression . Low expression of ZNF292 is associated with poor prognosis in certain cancers, such as esophageal squamous cell carcinoma (ESCC) .

These dual functions make ZNF292 a target of interest for both neurodevelopmental and cancer research.

What applications are ZNF292 antibodies suitable for?

ZNF292 antibodies have been validated for multiple research applications:

When selecting an antibody for a specific application, researchers should review the validation data provided by manufacturers and consider the epitope location, as different antibodies target different regions of this large protein .

What is known about ZNF292 expression patterns?

ZNF292 shows tissue-specific expression patterns:

• Brain expression: Highly expressed in the developing human brain, particularly in the cerebellum during the prenatal period .

• Differential expression in cancer: Higher expression in adjacent normal tissues compared to tumor tissues, particularly in ESCC .

• Cellular expression: The specific cell types expressing ZNF292 in the brain, particularly in the cerebellar cortex and hippocampus, remain an active research question .

Understanding these expression patterns is crucial for experimental design, especially when selecting appropriate cellular models or tissue samples for ZNF292 research.

How should I validate ZNF292 antibody specificity for my experimental system?

Validating antibody specificity for ZNF292 requires a multi-step approach:

• Knockdown/knockout validation: Use siRNA or CRISPR/Cas9 to reduce or eliminate ZNF292 expression, then confirm reduced signal with the antibody.

• Overexpression validation: Express tagged ZNF292 (e.g., with GFP or FLAG) and confirm co-localization with antibody staining, or detection of the overexpressed protein by Western blot.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm signal reduction.

• Cross-reactivity assessment: Test the antibody in multiple species if cross-reactivity is claimed. Manufacturers report different cross-reactivity profiles:

  • Human-specific antibodies

  • Antibodies with predicted reactivity to other species (pig, bovine, sheep, rabbit, dog, chicken)

  • Antibodies with documented reactivity to multiple species (human, dog, cow, rabbit, guinea pig, horse, rat)

• Multiple antibody approach: Use antibodies targeting different epitopes of ZNF292 to confirm consistent results.

This validation is particularly important for ZNF292 as the protein is large (305 kDa) and may have multiple isoforms or post-translational modifications.

How can I optimize detection of ZNF292 in Western blots given its high molecular weight?

Detecting high molecular weight proteins like ZNF292 (~305 kDa) by Western blot presents several challenges. Follow these optimization strategies:

• Gel preparation and transfer optimization:

  • Use low percentage (6-7%) SDS-PAGE gels to better resolve high molecular weight proteins

  • Extend running time at lower voltage

  • Perform overnight transfer at low current (30-40 mA) at 4°C

  • Consider using specialized transfer systems designed for high molecular weight proteins

• Sample preparation modifications:

  • Use strong lysis buffers containing SDS and protease inhibitors

  • Avoid excessive heating of samples (65°C for 5-10 minutes instead of 95°C)

  • Include reducing agents (DTT or β-mercaptoethanol) at appropriate concentrations

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) substrates designed for high sensitivity

  • Consider longer exposure times than standard proteins

  • Use fresh antibody dilutions and optimize concentration (start with manufacturer recommendations, typically 1:500-1:1000)

• Controls:

  • Include positive controls where ZNF292 is known to be expressed

  • Consider using ZNF292-overexpressing cell lines as positive controls

  • Include molecular weight markers that extend to >250 kDa

Successful detection has been demonstrated in Jurkat whole cell lysates using immunoprecipitation followed by Western blot at 1μg/ml antibody concentration .

What is known about ZNF292 protein-protein interactions and how can antibodies help characterize them?

ZNF292 protein-protein interactions remain an active area of investigation:

• Known interaction partners:

  • ZNF292 has been proposed to interact with POU1F1, a member of the POU family of transcription factors

  • Potential interactions with members of the HP-1 complex have been suggested

  • No direct interactions have been clearly demonstrated

• Methodological approaches using antibodies:

  • Co-immunoprecipitation (Co-IP): Use anti-ZNF292 antibodies to pull down the protein complex and identify interaction partners by mass spectrometry or Western blot

  • Proximity ligation assay (PLA): Detect protein-protein interactions in situ using antibodies against ZNF292 and potential interaction partners

  • ChIP-seq: Identify DNA binding sites and potential co-factors using anti-ZNF292 antibodies

  • Bio-ID or APEX2 proximity labeling: Tag ZNF292 with a biotin ligase to identify proximal proteins, then use anti-ZNF292 antibodies to confirm localization

• Research limitations and considerations:

  • Previous ChIP-seq experiments with GFP-conjugated ZNF292 in HEK cells showed limited reproducibility between experimental trials

  • Native protein with anti-ZNF292 antibodies in relevant cell lines with higher ZNF292 expression might yield more consistent results

  • Consider epitope masking effects when ZNF292 is in complex with other proteins

How can ZNF292 antibodies be used to study its role in neurodevelopmental disorders?

Given the established link between ZNF292 variants and neurodevelopmental disorders , antibodies can be powerful tools for mechanistic studies:

• Tissue and cellular expression analyses:

  • Use IHC/IF with anti-ZNF292 antibodies to examine expression patterns in brain tissues from control and disorder models

  • Compare ZNF292 expression across developmental timepoints to understand temporal dynamics

  • Identify specific neural cell types expressing ZNF292 through co-staining with cell-type markers

• Patient-derived models:

  • Use Western blot and IF to analyze ZNF292 protein levels and localization in patient-derived cells (fibroblasts, iPSCs, neural organoids)

  • Compare wild-type and mutant ZNF292 behavior using antibodies that recognize epitopes outside the mutation sites

• Functional studies:

  • Use ChIP-seq with anti-ZNF292 antibodies to identify genomic binding sites and potential gene targets in neural cells

  • Compare DNA binding profiles between wild-type and mutant ZNF292 proteins

  • Analyze post-translational modifications of ZNF292 in the context of neurodevelopment using modification-specific antibodies

• Animal models:

  • Validate ZNF292 knockdown/knockout in animal models using antibody-based detection methods

  • Compare neural development and behavior with ZNF292 expression patterns in these models

How should I select the appropriate ZNF292 antibody for my research?

Selection of the optimal ZNF292 antibody requires consideration of multiple factors:

• Epitope location: Different antibodies target distinct regions of ZNF292:

  • Internal region antibodies

  • C-terminal antibodies

  • Antibodies targeting specific amino acid ranges (AA 2000-2050, AA 398-447, AA 1311-1360)

• Application specificity: Ensure the antibody is validated for your specific application with clear, published evidence:

  • Immunoprecipitation data shows successful detection of ZNF292 in Jurkat cell lysates

  • Western blot validation with appropriate controls

  • IHC/IF with appropriate positive and negative controls

• Host species compatibility: Consider the host species (most ZNF292 antibodies are rabbit polyclonal ) in relation to other antibodies used in multi-labeling experiments

• Validation rigor: Evaluate the extent of validation provided by manufacturers:

  • Specific detection of endogenous ZNF292

  • Validation across multiple applications

  • Published literature citing the specific antibody

• Lot-to-lot consistency: Request information on lot-to-lot validation, especially for polyclonal antibodies

What are the challenges in studying ZNF292 splice variants and how can antibodies help address them?

ZNF292 splicing complexity presents research challenges:

• Current knowledge of ZNF292 transcripts:

  • GRCh38 shows ZNF292 composed of eight exons, with the last exon being much larger and containing all 16 zinc fingers

  • GTEx Portal indicates 14 possible exons, but only the eight identified in GRCh38 show significant expression levels

  • The final exon shows expression levels twice as high as other expressed exons, suggesting alternate splicing

  • A computed transcript (exons 6-8) may correspond to a smaller protein variant

• Antibody-based approaches to studying splice variants:

  • Epitope-specific antibodies: Use antibodies targeting different regions to detect specific isoforms

  • Western blot analysis: Detect multiple bands corresponding to different isoforms

  • Immunoprecipitation followed by mass spectrometry: Identify protein isoforms expressed in specific tissues/cells

• Experimental design considerations:

  • Combine antibody-based protein detection with RT-PCR to correlate transcript and protein expression

  • Use isoform-specific siRNAs to selectively deplete specific variants and confirm antibody specificity

  • Consider that early studies used a recombinant protein from cDNA libraries that included only the last exon

• Research questions to address:

  • Is the full-length transcript translated and/or are there shorter alternately spliced transcripts?

  • Is the protein post-translationally cleaved?

  • Do different isoforms have distinct cellular functions or localization patterns?

How can ZNF292 antibodies be used to study its DNA binding and transcriptional regulation functions?

ZNF292 functions as a transcription factor with DNA binding capability. Antibodies can help characterize these functions:

• ChIP-seq approaches:

  • Previous attempts using GFP-conjugated ZNF292 in HEK cells showed limited reproducibility

  • Recommendations for improved ChIP-seq:

    • Use native protein with anti-ZNF292 antibodies

    • Select cell lines with higher ZNF292 expression

    • Optimize crosslinking and sonication conditions for this large protein

    • Validate antibody specificity for ChIP applications

• DNA binding characterization:

  • Early research found ZNF292 zinc fingers 10-12 bound to regions in the growth hormone promoter

  • Strong binding to the TRE at the promoter of MYH7 was also reported

  • These experiments used rat protein and oligonucleotides

  • Reproduce experiments with human ZNF292 and relevant oligonucleotides

• Transcriptional regulation studies:

  • Recent research revealed ZNF292 suppresses SKP2 expression at the transcriptional level in ESCC cells

  • ZNF292 binds to the promoter region of SKP2, as confirmed by ChIP assays

  • Luciferase reporter assays showed increased activity upon ZNF292 depletion, indicating its suppressive role

• Novel research directions:

  • Genome-wide identification of ZNF292 binding sites in relevant cell types (neural cells, cancer cell lines)

  • Characterization of transcriptional activator vs. repressor functions in different genomic contexts

  • Investigation of co-factors that modulate ZNF292 binding and function

What technical considerations are important when using ZNF292 antibodies for different cell and tissue types?

Optimizing ZNF292 antibody protocols across different experimental systems requires careful attention to several factors:

• Expression level variations:

  • ZNF292 is highly expressed in the developing human brain, particularly the cerebellum

  • Expression levels vary between normal and cancer tissues, with higher expression in normal tissues

  • Adjust antibody dilutions based on expected expression levels (lower dilutions for low-expressing samples)

• Tissue-specific optimization:

  • Brain tissue considerations:

    • Optimize fixation conditions (4% PFA, shorter fixation times)

    • Consider antigen retrieval methods (heat-mediated in citrate buffer has been successful)

    • Use appropriate permeabilization for this nuclear protein

  • Cancer tissue considerations:

    • Evaluate expression in paired normal/tumor samples

    • Consider background issues in highly vascularized tumors

    • Validate specificity with appropriate controls

• Cell line selection:

  • Jurkat cells have been successfully used for ZNF292 immunoprecipitation

  • KYSE30 cells have been used in ZNF292 functional studies

  • Consider endogenous expression levels when selecting cell models

• Protocol optimization by application:

  • IHC optimization:

    • Dilution range: 1:50-1:200

    • Antigen retrieval: Heat-mediated in citrate buffer

    • Blocking: 3% hydrogen peroxide followed by protein blocking

    • Detection systems: Diaminobenzidine as chromogenic substrate

  • IF/ICC optimization:

    • Dilution range: 1:100-1:500

    • Fixation: 4% PFA (10-15 minutes)

    • Permeabilization: 0.1-0.3% Triton X-100

    • Mounting media with DAPI for nuclear counterstain

How can ZNF292 antibodies contribute to understanding its roles in cancer biology?

ZNF292 has been identified as a tumor suppressor, and antibodies can help elucidate its mechanisms:

• Expression pattern analysis:

  • ZNF292 is highly expressed in adjacent and normal tissues compared to tumor tissues in ESCC

  • Immunohistochemistry with ZNF292 antibodies can help establish expression patterns across different cancer types

  • Correlate expression levels with clinical outcomes and prognostic indicators

• Mechanistic studies:

  • ZNF292 depletion promotes cell cycle progression by activating the SKP2/P27 signaling pathway

  • Western blot analysis with phospho-specific antibodies can track changes in this pathway

  • ZNF292 antibodies can help identify additional regulatory mechanisms through proteomics approaches

• Therapeutic implications:

  • Monitor ZNF292 expression changes in response to therapeutic interventions

  • Use antibodies to identify biomarkers associated with ZNF292 status

  • Explore ZNF292 as a potential therapeutic target in cancers where its expression is altered

• Research gaps to address:

  • Relationship between ZNF292 mutations/variants and protein function in cancer contexts

  • Tissue-specific roles of ZNF292 in different cancer types

  • Potential for ZNF292 status as a diagnostic or prognostic marker

What epigenetic functions might ZNF292 have and how can antibodies help investigate them?

Emerging evidence suggests ZNF292 may have epigenetic functions:

• Relationship to RLF (its closest paralog):

  • RLF maintains CpG shores hypomethylated in a subset of promoters

  • ZNF292's N-terminal is highly similar to RLF

  • Investigate whether ZNF292 has similar functions in maintaining methylation patterns

• Research approaches using antibodies:

  • ChIP-seq combined with bisulfite sequencing: Identify correlations between ZNF292 binding and DNA methylation patterns

  • Co-IP followed by mass spectrometry: Identify interactions with chromatin modifying enzymes

  • Sequential ChIP (Re-ChIP): Determine co-occupancy with histone marks or other epigenetic regulators

• Cell-type maintenance:

  • Investigate ZNF292's potential role in maintaining cell-type specific gene expression patterns

  • Use antibodies to track ZNF292 localization during cell differentiation

  • Combine with transcriptomic and epigenomic profiling to identify regulatory networks

• Developmental dynamics:

  • Track changes in ZNF292 binding patterns across developmental timepoints

  • Correlate with changes in chromatin accessibility and histone modifications

  • Focus on brain development given ZNF292's role in neurodevelopmental disorders