ZNF408 Antibody

What is ZNF408 and what are its primary biological functions?

ZNF408 (Zinc Finger Protein 408) is a 95 kDa protein belonging to the zinc finger protein family, primarily involved in transcriptional regulation . It encodes a transcription factor of 720 amino acids that belongs to the class of C2H2 zinc finger proteins and is predicted to contain a SET (su(var)3–9, enhancer-of-zeste, trithorax) domain, which likely mediates protein-protein interactions in chromatin-regulated gene expression . The protein contains ten C2H2-type zinc finger binding domains that are crucial for DNA binding . ZNF408 plays a significant role in retinal vasculature development, as demonstrated through zebrafish models, and mutations in this gene have been linked to familial exudative vitreoretinopathy (FEVR) .

Which experimental applications are ZNF408 antibodies validated for?

Based on the available research data, ZNF408 antibodies have been validated for immunohistochemistry of paraffin-embedded tissues (IHC-P) . Specific applications include:

• Detection of ZNF408 in human tissue samples, particularly testis tissue

• Immunostaining of zebrafish cryosections at 5 days post-fertilization (dpf) using anti-znf408 antibody (1:100 dilution)

• Subcellular localization studies in cell culture systems to determine nuclear versus cytoplasmic distribution

The rabbit polyclonal ZNF408 antibody has been confirmed to react with human samples, using an immunogen corresponding to a recombinant fragment within human ZNF408 amino acids 300-650 .

What are the recommended protocols for ZNF408 immunohistochemistry in paraffin-embedded tissues?

For optimal IHC-P results with ZNF408 antibodies, researchers should:

• Process tissue samples through standard paraffin embedding procedures

• Section tissues at appropriate thickness (typically 4-7 μm)

• Deparaffinize and rehydrate sections following standard protocols

• Perform antigen retrieval as recommended by the antibody manufacturer

• Block with appropriate blocking solution to minimize background staining

• Apply ZNF408 antibody at a 1:100 dilution (based on validated protocols)

• Incubate overnight at 4°C or as specified in the protocol

• Apply appropriate secondary antibody system and visualization method

• Counterstain, dehydrate and mount as per standard procedures

In published research, this methodology has successfully visualized ZNF408 in human testis tissue sections .

How can ZNF408 antibodies be used to investigate subcellular protein localization in wild-type versus mutant variants?

ZNF408 antibodies are valuable tools for studying the differential subcellular localization of wild-type versus mutant proteins, particularly in the context of disease-associated mutations. Research protocols should include:

• Cell culture and transfection setup:

  • Transiently transfect appropriate cell lines (e.g., COS-1) with constructs expressing HA-tagged fusion proteins of wild-type ZNF408 or mutant variants (e.g., ZNF408_Ser126Asn, ZNF408_His455Tyr)

  • Include separate transfections for wild-type, each mutant variant, and co-transfections of wild-type with mutant variants

• Immunocytochemical analysis:

  • Fix cells at 24-48 hours post-transfection

  • Permeabilize appropriately to allow antibody access to nuclear and cytoplasmic compartments

  • Use anti-ZNF408 antibody in combination with anti-tag antibodies to distinguish between transfected and endogenous proteins

  • Apply fluorescent secondary antibodies for visualization and include nuclear counterstain (DAPI)

• Analysis parameters:

  • Quantify the nuclear versus cytoplasmic distribution

  • For co-transfection experiments, assess potential dominant-negative effects where mutant protein may affect wild-type localization

Research using this approach has demonstrated that wild-type ZNF408 and the p.Ser126Asn mutant localize almost exclusively to the nucleus, while the p.His455Tyr mutant is predominantly cytoplasmic . Furthermore, co-transfection experiments revealed that the p.His455Tyr mutant can retain wild-type ZNF408 in the cytoplasm, suggesting oligomerization and a dominant-negative effect .

What methodological considerations are important when using ZNF408 antibodies in zebrafish models?

When utilizing ZNF408 antibodies in zebrafish models, researchers should consider:

• Fixation and sectioning protocols:

  • Incubate larvae (typically 5 dpf) in 10% (w/v) sucrose/PBS solution for 10 minutes for cryoprotection

  • Embed in optimal cutting temperature reagent and snap freeze

  • Prepare cryosections at approximately 7 μm thickness

• Immunostaining procedure:

  • Fix sections in 4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilize with 0.01% (v/v) Tween 20/PBS (20 minutes)

  • Block using 10% normal goat serum and 2% BSA (1 hour)

  • Apply rabbit anti-znf408 antibody at 1:100 dilution overnight

  • Use appropriate fluorescent secondary antibodies (e.g., goat-anti-rabbit Alexa Fluor 568 at 1:800 dilution)

  • Counterstain with DAPI and mount using appropriate mounting medium

• Image acquisition and analysis:

  • Capture images using high-resolution fluorescence microscopy

  • Analyze multiple sections (minimum three) for relevant parameters

  • Use appropriate software (e.g., ZEN3.0) for quantitative analysis

This approach has been successfully employed to study znf408 expression and function in zebrafish models of FEVR .

How can researchers utilize ZNF408 antibodies to correlate genotype with protein expression and localization in disease models?

To correlate ZNF408 genotype with protein expression and localization patterns in disease models:

• Comprehensive experimental design:

  • Generate or obtain relevant disease models (cell lines, animal models) with specific ZNF408 mutations

  • Include appropriate controls (wild-type, heterozygous, and homozygous mutants when possible)

  • Design experiments to capture both protein expression levels and subcellular distribution

• Multi-method validation approach:

  • Combine immunoblotting to quantify total protein levels

  • Use immunohistochemistry/immunofluorescence to assess tissue distribution and subcellular localization

  • Consider co-staining with markers of relevant subcellular compartments and interacting proteins

• Functional correlation analysis:

  • Link protein expression/localization data with functional readouts (e.g., vasculature development in zebrafish)

  • Assess whether protein mislocalization correlates with phenotype severity

Research has shown that mutations such as p.His455Tyr affect the nuclear localization of ZNF408, with the mutant protein predominantly localizing to the cytoplasm rather than the nucleus . This mislocalization correlates with vascular defects observed in both patient samples and animal models, supporting a pathogenic mechanism involving disrupted nuclear function of ZNF408 .

What strategies can resolve non-specific binding when using ZNF408 antibodies?

When encountering non-specific binding during ZNF408 antibody applications, researchers should implement these optimization strategies:

• Antibody dilution optimization:

  • Test a range of dilutions (e.g., 1:50 to 1:500) based on the validated 1:100 dilution reported in literature

  • Determine the optimal balance between specific signal and background

• Blocking protocol refinement:

  • Increase blocking solution concentration or time (e.g., use 10% normal serum from the species of secondary antibody plus 2% BSA)

  • Consider alternative blocking agents if background persists

  • Implement additional blocking steps with species-specific Fab fragments if cross-reactivity is observed

• Tissue/sample preparation adjustments:

  • Optimize fixation conditions (time, temperature, fixative composition)

  • Adjust antigen retrieval methods for IHC-P applications

  • Ensure complete permeabilization for intracellular antigens while preserving tissue morphology

• Validation controls:

  • Include peptide competition assays to confirm specificity

  • Use tissue from knockout models as negative controls when available

  • Include isotype controls to identify non-specific binding of immunoglobulins

These strategies address the common sources of non-specific binding while maintaining the sensitivity required to detect ZNF408 in research applications.

How can researchers effectively validate ZNF408 antibodies for novel applications or species?

When validating ZNF408 antibodies for novel applications or species not previously tested, implement this systematic approach:

• Sequence homology analysis:

  • Align ZNF408 amino acid sequences between the immunogen source (human amino acids 300-650) and the target species

  • Assess the degree of conservation in the epitope region

  • Predict cross-reactivity based on percentage identity and conservation of key epitope residues

• Stepwise validation protocol:

  • Begin with Western blot to confirm detection of a protein of appropriate molecular weight (approximately 95 kDa)

  • Validate positive and negative controls (e.g., tissues known to express or not express ZNF408)

  • Include knockdown/knockout samples as definitive negative controls when possible

  • Progress to more complex applications (IHC, IF) only after initial validation

• Application-specific optimization:

  • For each new application, systematically optimize key parameters:

    • Sample preparation (fixation, antigen retrieval)

    • Antibody concentration

    • Incubation conditions (time, temperature, buffer composition)

    • Detection system parameters

• Functional validation:

  • Correlate antibody staining patterns with known biological functions

  • Confirm subcellular localization is consistent with expected transcription factor distribution (predominantly nuclear for wild-type)

This methodical approach ensures reliable results when extending ZNF408 antibody use to new experimental contexts.

How can ZNF408 antibodies be utilized to investigate its role in transcriptional regulation?

ZNF408 functions as a transcription factor with multiple zinc finger domains , and antibodies can help elucidate its regulatory activities through:

• Chromatin immunoprecipitation (ChIP) applications:

  • Optimize ChIP protocols using anti-ZNF408 antibodies to identify genomic binding sites

  • Perform ChIP followed by sequencing (ChIP-seq) to map genome-wide binding patterns

  • Compare wild-type and mutant ZNF408 binding profiles to identify dysregulated targets

  • Consider dual crosslinking approaches to capture potentially indirect DNA interactions

• Protein complex identification:

  • Employ immunoprecipitation with ZNF408 antibodies followed by mass spectrometry

  • Identify protein interaction partners in different cellular contexts and in response to stimuli

  • Focus on interactions mediated by the SET domain, which is involved in protein-protein interactions

• Transcriptional activity assessment:

  • Use ZNF408 antibodies in combination with antibodies against transcriptional machinery components

  • Perform sequential ChIP (re-ChIP) to determine co-occupancy with other transcription factors

  • Correlate ZNF408 binding with active or repressive chromatin marks through co-staining approaches

• Functional genomics integration:

  • Combine ZNF408 binding data with transcriptome analysis after ZNF408 manipulation

  • Identify direct versus indirect transcriptional targets

  • Establish the gene regulatory networks controlled by ZNF408, particularly in vascular development contexts

These approaches would help identify the transcriptional targets of ZNF408, which could reveal other genes important for retinal angiogenesis and potential candidates for mutations in related disorders .

What is the significance of ZNF408 mutations in familial exudative vitreoretinopathy (FEVR) and how can antibodies help characterize their effects?

ZNF408 mutations, particularly p.His455Tyr, have been implicated in familial exudative vitreoretinopathy (FEVR) . Antibodies can help characterize these mutations through:

• Patient sample analysis:

  • Analyze ZNF408 expression and localization in available patient-derived samples

  • Compare protein patterns between affected and unaffected family members

  • Correlate antibody staining patterns with disease severity and progression

• Mutation-specific functional characterization:

  • Use site-directed mutagenesis to introduce specific FEVR-associated mutations (e.g., p.His455Tyr, p.Ser126Asn)

  • Assess mutant protein stability, localization, and function using antibody-based assays

  • Identify differential binding partners that may explain pathogenic mechanisms

• Dominant-negative effect quantification:

  • Develop quantitative co-localization assays to measure the degree to which mutant ZNF408 sequesters wild-type protein in the cytoplasm

  • Establish dose-response relationships between mutant and wild-type protein ratios

  • Correlate sequestration efficiency with functional outcomes in model systems

• Therapeutic target identification:

  • Use antibodies to screen for compounds that rescue normal ZNF408 localization

  • Develop high-content screening assays based on ZNF408 antibody staining

  • Monitor potential therapeutic interventions in disease models

Research has demonstrated that the p.His455Tyr mutation acts in a dominant-negative fashion by retaining the wild-type ZNF408 protein in the cytoplasm, preventing its normal nuclear localization and function . This mechanism helps explain the autosomal dominant inheritance pattern observed in FEVR families with ZNF408 mutations.

How can ZNF408 antibodies be employed in comparative studies across different model organisms?

ZNF408 antibodies can facilitate comparative studies across different experimental models through:

• Cross-species protocol adaptations:

  • Modify immunostaining protocols for different model systems (cell lines, zebrafish, mouse models)

  • Adjust fixation and permeabilization conditions based on tissue type and preservation requirements

  • Optimize antibody concentration for each model system

• Evolutionarily conserved function assessment:

  • Compare subcellular localization patterns across species to identify conserved mechanisms

  • Assess ZNF408 expression during development in different organisms

  • Correlate expression patterns with conserved developmental processes, particularly vascular development

• Mutation modeling across species:

  • Generate equivalent mutations (e.g., p.His455Tyr) in different model organisms

  • Use antibodies to compare wild-type and mutant protein behaviors across species

  • Identify species-specific differences in ZNF408 function or regulation

Research has demonstrated that knockdown of znf408 in zebrafish results in defects in developing retinal and trunk vasculature, which can be rescued by coinjection of human wild-type ZNF408 RNA but not by p.His455Tyr mutant ZNF408 RNA . This highlights the evolutionary conservation of ZNF408 function in vascular development and validates zebrafish as a model for studying FEVR.

What methodological considerations are important when using ZNF408 antibodies in CRISPR/Cas9-generated model systems?

When working with CRISPR/Cas9-generated ZNF408 mutant models, researchers should consider:

• Mutation validation strategy:

  • Use ZNF408 antibodies to confirm protein alterations at the protein level

  • Verify that introduced mutations result in the expected protein changes (truncation, mislocalization)

  • Implement Western blotting to assess protein size changes and immunostaining to evaluate localization patterns

• Mosaic expression analysis:

  • Develop protocols to distinguish between mutant and wild-type cells in mosaic animals

  • Consider double-staining approaches with markers of successful CRISPR editing

  • Quantify the percentage of cells with altered ZNF408 expression or localization

• Off-target effect assessment:

  • Use ZNF408 antibodies to evaluate protein expression in tissues not expected to be affected

  • Compare multiple CRISPR-generated lines to distinguish mutation-specific from off-target effects

  • Implement rescue experiments with wild-type protein to confirm phenotype specificity

• Genotype-phenotype correlation:

  • Design experiments to correlate ZNF408 protein expression/localization with phenotypic outcomes

  • Compare homozygous and heterozygous mutants to understand gene dosage effects

  • Develop quantitative image analysis pipelines for consistent phenotype assessment

Researchers have successfully generated zebrafish models with frameshift mutations targeting the fourth zinc finger domain of znf408, designed to mimic the human p.His455Tyr mutation . These models provide valuable tools for studying ZNF408 function in vivo.