ZNF98 Antibody

What is ZNF98 and why is it significant for research?

ZNF98 (Zinc finger protein 98) is a human protein that likely functions as a transcriptional regulator. It belongs to the krueppel C2H2-type zinc-finger protein family, which plays important roles in gene expression regulation . Research significance stems from its:

• Potential role in gene transcription regulation

• Human-specific expression patterns

• Evolutionary significance as it appears to have evolved before the Neandertal-Denisovan split

• Enriched expression in neural progenitor cells of the fetal human neocortex, suggesting potential involvement in human brain development

Understanding ZNF98 function may provide insights into human-specific aspects of gene regulation and potentially neurodevelopment.

What applications are validated for commercially available ZNF98 antibodies?

ZNF98 antibodies have been validated for multiple research applications, with different antibodies showing specific application profiles:

When selecting an antibody for your research, consider which applications have been experimentally validated for each specific antibody clone or preparation.

What are the recommended sample preparation methods for ZNF98 antibody applications?

Based on validated protocols, the following sample preparation methods are recommended:

For Western blot:

• Use cell lysates (e.g., MDA-MB435 cell line has been successfully used)

• Load approximately 35μg protein per lane for optimal detection

• Standard SDS-PAGE and transfer protocols are applicable

For Immunohistochemistry:

• Both frozen sections and formalin-fixed paraffin-embedded (FFPE) tissues have been validated

• For FFPE sections, standard antigen retrieval methods are recommended

• Human lymph node tissue has been successfully used for validation

For Immunofluorescence:

• HeLa cells have been validated as suitable specimens

• Use 1:50-1:200 dilution of primary antibody

• Secondary detection with fluorophore-conjugated antibodies (e.g., Alexa Fluor 488-conjugated anti-rabbit IgG)

How should I optimize antibody dilutions for different ZNF98 detection methods?

Optimization should follow a systematic approach based on validated starting points:

For Western blot:

• Start with manufacturer's recommended dilution (typically 1:500-1:1000)

• Perform a dilution series if signal-to-noise ratio is suboptimal

• Consider longer primary antibody incubation (overnight at 4°C) for weaker signals

For Immunofluorescence:

• Begin with 1:100 dilution as validated for HeLa cells

• Test a range from 1:50 to 1:200 to determine optimal signal-to-background ratio

For ELISA:

• Start with 1:2000 dilution and optimize up to 1:10000 based on signal intensity

• Include proper negative controls to establish background levels

Remember that optimal dilutions may vary between different antibody lots and experimental conditions. Validation using positive and negative controls is essential for each new experimental setup.

What controls should be included when using ZNF98 antibodies?

A robust control strategy for ZNF98 antibody experiments should include:

Positive controls:

• Cell lines with known ZNF98 expression (e.g., MDA-MB435, HeLa cells)

• Human lymph node tissue for IHC applications

• Recombinant ZNF98 protein (available as fragment protein covering aa 11-261)

Negative controls:

• Primary antibody omission control

• Isotype control (rabbit or mouse IgG depending on the primary antibody host)

• Non-expressing tissues or cell lines (requires prior validation)

• For genetic approaches, CRISPR-edited cell lines with ZNF98 knockout can serve as definitive negative controls

Blocking peptide controls:

• When available, pre-incubation of the antibody with immunizing peptide should abolish specific signals

• The immunizing peptide for certain antibodies corresponds to amino acids 520-549 from the C-terminal region

What storage conditions maximize ZNF98 antibody stability and performance?

To maintain optimal antibody performance:

• Store antibodies at -20°C for long-term storage, avoiding freeze/thaw cycles

• For antibodies in glycerol formulations (e.g., those in 50% glycerol buffer), aliquot upon receipt to minimize freeze/thaw cycles

• Some antibodies can be stored at 4°C for short-term use (typically up to one month)

• Follow manufacturer-specific recommendations, as formulations vary:

  • 50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 (Epigentek)

  • PBS with 0.09% sodium azide (Invitrogen)

Antibody performance should be validated periodically, especially after prolonged storage or when using antibodies beyond the manufacturer's expiration date.

How can ZNF98 antibodies be utilized to study transcriptional regulation mechanisms?

As ZNF98 is implicated in transcriptional regulation, several advanced approaches can be employed:

Chromatin Immunoprecipitation (ChIP):

• Though not explicitly validated in the search results, antibodies suitable for immunoprecipitation (e.g., PCRP-ZNF98-1A11) may be adapted for ChIP applications

• Protocol modification suggestions:

  • Use crosslinking conditions optimized for zinc finger proteins (typically 1% formaldehyde for 10 minutes)

  • Include zinc in buffers (100μM ZnCl₂) to maintain zinc finger domain integrity

  • Consider dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde for improved capture

Co-Immunoprecipitation (Co-IP):

• Use ZNF98 antibodies to identify protein interaction partners involved in transcriptional complexes

• The mouse monoclonal antibody (PCRP-ZNF98-1A11) validated for immunoprecipitation would be the primary choice

Proximity Ligation Assay (PLA):

• Combine ZNF98 antibodies with antibodies against suspected interaction partners

• This technique can visualize protein-protein interactions in situ with high sensitivity

When interpreting results, consider the structural features of ZNF98, including its multiple C2H2-type zinc finger motifs that likely mediate DNA binding .

What approaches can be used to study ZNF98 in the context of human neocortical development?

Given ZNF98's enriched expression in neural progenitor cells of the fetal human neocortex , several specialized approaches can be implemented:

Immunohistochemistry of developmental tissue:

• Use validated antibodies for IHC-P on human fetal brain sections

• Employ double labeling with neural progenitor markers:

  • PAX6 (radial glial cells)

  • TBR2/EOMES (intermediate progenitors)

  • SOX2 (neural stem cells)

In vitro models:

• Human iPSC-derived cerebral organoids

  • Track ZNF98 expression during organoid development using IF or Western blot

  • Correlate with developmental timing and progenitor populations

Functional studies:

• Similar approaches to those used for other human-specific genes like NOTCH2NL

• NOTCH2NL (another human-specific gene with similar expression pattern) has been shown to promote basal progenitor proliferation in mice

• Consider expressing ZNF98 in mouse neocortex to study effects on progenitor behavior

Single-cell analysis:

• Apply antibodies in mass cytometry (CyTOF) or for immunofluorescence in single-cell Western blot platforms

• Correlate with transcriptomics data from existing neural progenitor databases

How might ZNF98 antibodies be used to investigate evolutionary aspects of human-specific genes?

ZNF98 represents an interesting case for evolutionary studies as it appears to be human-specific and evolved before the Neandertal-Denisovan split . Research approaches include:

Comparative analysis across species:

• Use antibodies with validated human reactivity to test cross-reactivity with tissues from:

  • Non-human primates (chimpanzee, gorilla, etc.)

  • Other mammals

• Lack of signal in non-human samples would support human-specific expression

Analysis of paralogs:

• ZNF492 is reported to be a chimera consisting of the original KRAB repressor domain and the acquired ZNF98 DNA binding domain

• Compare expression patterns of ZNF98 and ZNF492 using specific antibodies

• Investigate potential functional divergence between paralogs

Archaic human studies:

• Though challenging, collaboration with paleogenomics researchers could explore ZNF98 in ancient human samples

• Antibody-based approaches might include highly sensitive immunoassays on preserved proteins

Expression analysis in humanized models:

• Apply ZNF98 antibodies in humanized mouse models carrying human genomic regions

This evolutionary perspective is important as gene duplication events have contributed significantly to human-specific aspects of brain development .

What are common challenges when using ZNF98 antibodies and how can they be addressed?

Researchers working with ZNF98 antibodies may encounter several technical challenges:

High background in immunohistochemistry/immunofluorescence:

• Increase blocking stringency (5% BSA or 10% normal serum from secondary antibody host species)

• Optimize antibody dilution (try more dilute solutions)

• Ensure thorough washing (increase number and duration of wash steps)

• For paraffin sections, optimize antigen retrieval conditions

Weak or absent signal in Western blot:

• Confirm protein loading (35μg per lane has been validated for some applications)

• Try enhanced chemiluminescence (ECL) substrates with higher sensitivity

• Optimize transfer conditions for high molecular weight proteins

• Consider using PVDF membranes instead of nitrocellulose for potentially better protein retention

Multiple bands in Western blot:

• Potential explanation: detection of different isoforms or post-translational modifications

• Compare band pattern with predicted molecular weight (check UniProt entry A6NK75)

• Use recombinant ZNF98 protein (aa 11-261) as a positive control to identify specific band

Cross-reactivity concerns:

• ZNF98 has paralogs and belongs to a large protein family with similar domains

• Validate specificity using overexpression systems or knockout models

• Consider using multiple antibodies targeting different epitopes

How can I differentiate between ZNF98 and its close paralogs in experimental systems?

Distinguishing ZNF98 from related proteins requires careful experimental design:

Antibody selection strategy:

• Choose antibodies raised against unique regions of ZNF98

• The C-terminal region (aa 520-549) used for some antibodies may provide specificity

• Avoid antibodies targeting highly conserved zinc finger domains shared with paralogs

Validation approaches:

• Peptide competition assays using specific peptides from ZNF98 vs. paralogs

• siRNA/shRNA knockdown experiments with ZNF98-specific sequences

• Parallel detection with antibodies known to detect specific paralogs (e.g., ZNF492)

Data analysis considerations:

• When analyzing RNA-seq data alongside antibody-based experiments, filter for ZNF98-specific reads

• For mass spectrometry validation, identify peptides unique to ZNF98 that aren't shared with paralogs

Comparative expression analysis:

• ZNF492 has a chimeric structure incorporating elements from ZNF98

• Compare expression patterns between ZNF98 and ZNF492 in the same tissues/cells

What considerations are important when interpreting ZNF98 localization data from imaging studies?

When analyzing subcellular localization of ZNF98:

Expected localization pattern:

• As a putative transcription factor, ZNF98 is expected to show predominantly nuclear localization

• Immunofluorescence has confirmed nuclear localization in HeLa cells

• Consider that zinc finger proteins may show nucleolar exclusion or specific nuclear subdomains

Co-localization studies:

• Combine ZNF98 antibodies with markers for:

  • Nuclear compartments (e.g., SC35 for splicing speckles, fibrillarin for nucleoli)

  • Chromatin states (H3K27me3 for repressed regions, H3K4me3 for active promoters)

  • Other transcription factors that might function in similar pathways

Dynamic localization:

• Consider whether localization changes during:

  • Cell cycle phases (using markers like Ki67 or phospho-histone H3)

  • Differentiation processes (particularly relevant in neural progenitor studies)

  • Response to signaling or stress conditions

Technical considerations:

• Use super-resolution microscopy for detailed nuclear distribution patterns

• Consider live-cell imaging with fluorescently tagged antibody fragments for dynamic studies

• Validate observations with biochemical fractionation and Western blot analysis

How might ZNF98 antibodies contribute to understanding human-specific aspects of neurodevelopment?

Given ZNF98's enriched expression in human neural progenitor cells and its human-specific nature , antibody-based studies could reveal:

Developmental timing:

• Track expression during cortical development using tissue samples across gestational ages

• Correlate with neurogenesis waves and cortical layer formation

• Compare with other human-specific genes like NOTCH2NL that promote basal progenitor proliferation

Cell-type specificity:

• Determine precise neural progenitor subtypes expressing ZNF98:

  • Apical radial glia

  • Basal radial glia

  • Intermediate progenitors

• This fine mapping could reveal roles in specific neurogenic lineages

Functional significance:

• Use antibodies to identify ZNF98 binding partners unique to human neural development

• Apply in models where ZNF98 is ectopically expressed (similar to NOTCH2NL studies)

• Investigate potential roles in:

  • Progenitor proliferation vs. differentiation

  • Neuronal migration

  • Cortical folding mechanisms

Disease relevance:

• Explore ZNF98 expression in neurodevelopmental disorders

• Given that some NBPF genes containing DUF1220 domains have been implicated in disorders like microcephaly, macrocephaly, autism, and schizophrenia , ZNF98 might have similar clinical relevance

What is the potential for combining ZNF98 antibodies with cutting-edge single-cell technologies?

Integration of ZNF98 antibodies with emerging single-cell methodologies offers powerful research opportunities:

Single-cell Western blotting:

• Detect ZNF98 in individual cells to quantify expression heterogeneity

• Correlate with cell morphology and other markers

Imaging mass cytometry:

• Include ZNF98 antibodies in antibody panels for CyTOF imaging

• Simultaneously detect dozens of proteins in tissue sections

• Create high-dimensional maps of ZNF98 expression relative to cell states

CITE-seq approaches:

• Utilize oligonucleotide-tagged ZNF98 antibodies

• Simultaneously capture surface protein and transcriptome information

• Correlate ZNF98 protein levels with gene expression profiles

Spatial transcriptomics with protein detection:

• Combine ZNF98 immunofluorescence with spatial transcriptomics

• Overlay protein localization with transcriptional signatures

• Particularly valuable for studying regional differences in developing neocortex

These technological combinations could reveal how ZNF98 expression varies across different neural progenitor populations and developmental zones, providing insight into its function in human-specific aspects of brain development.

How can structural insights inform the development and application of next-generation ZNF98 antibodies?

Understanding ZNF98's structure can guide more sophisticated antibody development:

Domain-specific antibodies:

• ZNF98 contains multiple C2H2 zinc finger domains

• Developing antibodies against specific functional domains could:

  • Distinguish between DNA-binding functions

  • Block specific protein-protein interactions

  • Target conformation-specific epitopes

Conformational antibodies:

• Design antibodies that recognize specific zinc finger protein conformations

• These could distinguish between DNA-bound and unbound states

• Useful for studying regulatory dynamics in different cellular contexts

Structure-based epitope selection:

• As structural information becomes available, select epitopes that:

  • Are accessible in native protein

  • Don't interfere with critical functional interactions

  • Provide maximum specificity against paralogs

  • Work in multiple applications without denaturation requirements

Application-optimized antibodies:

• Develop antibodies specifically optimized for:

  • Live-cell imaging (non-toxic, membrane-permeable formats)

  • Super-resolution microscopy (with appropriate fluorophores)

  • Proximity labeling approaches (engineered for APEX or BioID fusion)

As research on zinc finger proteins continues to advance, these structure-informed approaches will enable more precise interrogation of ZNF98's functions in transcriptional regulation and human development.