ZNF114 Antibody

What is ZNF114 and what are its primary functions in cellular processes?

ZNF114 is a zinc finger protein encoded by the ZNF114 gene located on chromosome 19q13.33 . This protein is characterized by its zinc finger domains that enable identical protein binding activity and is primarily involved in the regulation of DNA-templated transcription . ZNF114 is found in extracellular exosomes and participates in transcriptional networks, though its precise regulatory mechanisms remain under investigation.

The protein belongs to the broader zinc-finger protein (ZFP) family, which has been implicated in numerous cellular processes including transcriptional regulation and RNA binding . Recent multi-omics analyses have revealed that many ZFPs, including those structurally similar to ZNF114, have multifunctional roles beyond their initially characterized functions .

What techniques can ZNF114 antibodies be effectively used for in research protocols?

ZNF114 antibodies have demonstrated utility in several key research techniques:

The Prestige Antibodies line of ZNF114 antibodies has been extensively validated for immunohistochemistry applications at dilutions of 1:200-1:500 . These antibodies have been tested across 44 normal human tissues and 20 common cancer tissue types, providing comprehensive validation data accessible through the Human Protein Atlas . For optimal results, researchers should validate each specific antibody lot for their particular application and cell/tissue system.

How should researchers validate ZNF114 antibodies before experimental use?

Validation of ZNF114 antibodies should follow these methodological steps:

• Positive control testing: Use tissues or cell lines known to express ZNF114, as documented in the Human Protein Atlas.

• Specificity confirmation: Perform blocking experiments using recombinant ZNF114 protein controls. For instance, the Human ZNF114 (aa 144-217) Control Fragment Recombinant Protein can be used for blocking experiments with corresponding antibodies . Pre-incubate the antibody-protein control fragment mixture at a 100x molar excess based on concentration and molecular weight for 30 minutes at room temperature before application .

• Knockdown/knockout verification: If possible, test antibody reactivity in ZNF114 knockdown/knockout systems to confirm specificity.

• Cross-reactivity assessment: Evaluate potential cross-reactivity with other zinc finger proteins, particularly those with sequence homology to ZNF114.

• Application-specific validation: Optimize conditions specifically for your experimental system and technique (IHC, Western blot, etc.).

What are the optimal sample preparation conditions for ZNF114 immunohistochemistry?

For optimal ZNF114 detection in immunohistochemistry applications:

• Fixation: Standard formalin fixation (10% neutral buffered formalin for 24-48 hours) is generally suitable for ZNF114 antibody applications in tissue samples.

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended, though some researchers may find EDTA buffer (pH 9.0) more effective depending on tissue type.

• Blocking: Use 5-10% normal serum from the species in which the secondary antibody was raised, supplemented with 1% BSA to reduce background.

• Antibody incubation: Apply the ZNF114 primary antibody (e.g., HPA029553) at a dilution of 1:200-1:500 for optimal staining . Incubate overnight at 4°C for best results.

• Detection system: Use a polymer-based detection system compatible with rabbit primary antibodies since most ZNF114 antibodies are rabbit-derived .

This protocol has been extensively validated through the Human Protein Atlas project, which has tested ZNF114 antibodies across numerous normal and disease tissues .

How can researchers optimize Western blotting protocols for ZNF114 detection?

For optimal Western blot detection of ZNF114:

• Protein extraction: Use RIPA buffer supplemented with protease inhibitors, as ZNF114 is potentially subject to proteolytic degradation.

• Sample preparation: Heat samples at 95°C for 5 minutes in reducing sample buffer containing SDS and DTT.

• Gel selection: Use 10-12% polyacrylamide gels, as ZNF114 has a molecular weight that falls within this separation range.

• Transfer conditions: Transfer at 100V for 1 hour using a wet transfer system with methanol-containing transfer buffer for optimal protein transfer.

• Blocking: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Antibody incubation: Dilute primary ZNF114 antibody according to manufacturer recommendations and incubate overnight at 4°C. For polyclonal antibodies like HPA029553, start with a 1:1000 dilution and adjust as needed .

• Verification: Include both positive controls and molecular weight markers to confirm specificity.

Positive Controls:

• Recombinant protein: Human ZNF114 (aa 144-217) Control Fragment Recombinant Protein can serve as a positive control .

• Cell lines: Based on Human Protein Atlas data, select cell lines with confirmed ZNF114 expression.

• Tissue samples: Refer to the Human Protein Atlas for tissues with documented ZNF114 expression.

Negative Controls:

• Antibody controls: Pre-incubation of the ZNF114 antibody with excess recombinant ZNF114 protein should abolish specific staining .

• Isotype controls: Use a non-specific IgG from the same species and at the same concentration as the ZNF114 antibody.

• Knockout/knockdown samples: If available, ZNF114 knockout or knockdown samples provide excellent negative controls.

For blocking experiments specifically, pre-incubate the antibody with a 100x molar excess of the protein control fragment for 30 minutes at room temperature before applying to your experimental system .

How does ZNF114 expression vary across different tissue types and disease states?

Based on the Human Protein Atlas project, which has characterized ZNF114 expression across 44 normal human tissues and 20 common cancer types, patterns of tissue-specific expression have been documented . While the provided search results don't provide comprehensive tissue expression data for ZNF114 specifically, the antibody product information notes that the expression patterns can be viewed through the Human Protein Atlas portal .

The connection between ZNF114 and disease states can be inferred from disease ontology enrichment analysis of zinc finger proteins. Research has shown that knockdown of 52 different ZFPs led to dysregulation of genes involved in at least one human disease . Although ZNF114 is not specifically mentioned in this context, this suggests potential involvement of ZNF114 in disease pathways, warranting further investigation.

What are common troubleshooting issues when using ZNF114 antibodies and how can they be resolved?

For ZNF114 specifically, researchers should note that as a zinc finger protein, it shares structural domains with other members of this large protein family. The polyclonal nature of available ZNF114 antibodies like HPA029553 may increase the risk of cross-reactivity, making proper validation especially important.

How do post-translational modifications affect ZNF114 function and antibody recognition?

While the search results don't provide specific information about post-translational modifications (PTMs) of ZNF114, research on other zinc finger proteins suggests that PTMs likely play an important role in regulating ZNF114 function. Common PTMs in zinc finger proteins include:

• Phosphorylation: Can alter DNA binding affinity and protein-protein interactions

• SUMOylation: Often affects subcellular localization and stability

• Ubiquitination: Regulates protein turnover and degradation

• Acetylation: Modifies DNA binding properties

The ZNF114 antibody product information indicates target "unmodified" post-translational modification status , suggesting that the antibody recognizes the unmodified form of the protein. Researchers investigating PTMs of ZNF114 should consider using PTM-specific antibodies or mass spectrometry-based approaches to supplement studies with general ZNF114 antibodies.

What is the role of ZNF114 in RNA binding and processing?

Recent research on zinc finger proteins has revealed that many ZFPs, beyond their classical role as transcription factors, have RNA-binding capabilities . While ZNF114 is not specifically highlighted for RNA binding in the provided search results, the integrated multi-omics analysis of zinc-finger proteins indicated that many ZFPs have RNA-binding domains (RBDs) that often overlap with intrinsically disordered regions (IDRs), zinc finger domains, and arginine-rich motif (ARM)-like domains .

Of the C2H2-containing ZFPs analyzed, 4 out of 13 contained putative RBDs that overlapped with C2H2 ZF domains, though in all cases, the RBDs also overlapped with other domain types . This suggests that while ZNF114 may potentially bind RNA, its ZF domains might not be the sole RNA-binding domain, with IDRs and ARM-like domains potentially contributing to RNA binding.

Researchers interested in ZNF114's potential RNA-binding capabilities should consider:

• RNA immunoprecipitation followed by sequencing (RIP-seq)

• Cross-linking immunoprecipitation (CLIP) methods

• In vitro RNA binding assays with recombinant ZNF114

What methods can researchers use to study ZNF114 protein-protein interactions?

To investigate protein-protein interactions involving ZNF114, researchers can employ several methodological approaches:

• Co-immunoprecipitation (Co-IP): Using ZNF114 antibodies to pull down protein complexes, followed by mass spectrometry or Western blotting to identify interaction partners.

• Proximity labeling methods: BioID or APEX2 fused to ZNF114 to identify proximal proteins in living cells.

• Yeast two-hybrid screening: Using ZNF114 as bait to identify potential interaction partners.

• Pull-down assays: Using recombinant ZNF114 protein as bait to capture interaction partners from cell lysates.

• Fluorescence resonance energy transfer (FRET): To study direct protein-protein interactions in living cells.

The search results indicate that ZNF114 enables identical protein binding activity , suggesting it may form homodimers or interact with other proteins. Recent multi-omics analysis of zinc finger proteins revealed extensive protein-protein interaction networks involving ZFPs , providing a methodological framework for studying ZNF114 interactions.

How can researcher accurately quantify ZNF114 expression levels in experimental samples?

For accurate quantification of ZNF114 expression, researchers should consider multiple complementary approaches:

Protein Level Quantification:

• Western blotting: Using validated ZNF114 antibodies with appropriate loading controls. Densitometric analysis should be performed across multiple biological replicates.

• ELISA: Development or use of commercial ELISA kits specific for ZNF114.

• Mass spectrometry: Label-free or isotope-labeled quantitative proteomics for absolute quantification.

mRNA Level Quantification:

• RT-qPCR: Design specific primers for ZNF114 with validation using standard curves and melt curve analysis.

• RNA-seq: For transcriptome-wide analysis including ZNF114 expression.

• Droplet digital PCR (ddPCR): For absolute quantification of ZNF114 transcript numbers.

For immunohistochemical quantification, researchers should follow standardized scoring systems such as H-score or Allred score, especially when examining ZNF114 expression across different tissue samples or disease states as documented in the Human Protein Atlas .

What is known about the genomic regulation of ZNF114 and how can researchers study it?

ZNF114 is located on chromosome 19q13.33 and contains 9 exons based on current genomic annotations . To study the genomic regulation of ZNF114, researchers can employ several approaches:

• Promoter analysis: Identify the promoter region of ZNF114 and characterize transcription factor binding sites using chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-seq).

• Epigenetic regulation: Analyze DNA methylation patterns and histone modifications in the ZNF114 locus using methylation-specific PCR, bisulfite sequencing, or ChIP-seq for histone marks.

• Enhancer identification: Use chromosome conformation capture techniques (3C, 4C, Hi-C) to identify distal regulatory elements that interact with the ZNF114 promoter.

• CRISPR-based approaches:

  • CRISPR interference (CRISPRi) to repress ZNF114 transcription

  • CRISPR activation (CRISPRa) to enhance ZNF114 expression

  • CRISPR-mediated deletion of potential regulatory elements

• Reporter assays: Clone potential regulatory regions upstream of reporter genes to assess their activity in regulating transcription.

Recent studies on zinc finger proteins have utilized CUT&RUNTools 2.0 for data analysis after adapter trimming with Trimmomatic (v0.36) , providing a methodological template for studying ZNF114 regulation.

How does ZNF114 expression correlate with disease progression in cancer studies?

While the search results don't provide specific data on ZNF114 in cancer progression, they do mention that the Human Protein Atlas includes data from the Cancer Atlas project, which has tested antibodies against 20 of the most common cancer types . This suggests that data on ZNF114 expression in various cancers may be available through this resource.

Researchers interested in studying ZNF114 in cancer should:

• Consult the Human Protein Atlas for baseline expression data across cancer types

• Perform immunohistochemical staining of tissue microarrays containing samples from different cancer stages to assess correlation with progression

• Analyze public gene expression databases (TCGA, GEO) for ZNF114 expression patterns across cancer types and stages

• Consider survival analysis to determine whether ZNF114 expression correlates with patient outcomes

It's worth noting that research on other zinc finger proteins has shown that knockdowns of various ZFPs led to differential expression of genes involved in human diseases, including cancers . This suggests that ZNF114 may similarly play roles in disease processes that warrant further investigation.

What are the current limitations in ZNF114 research and what methodological advances might overcome them?

Current limitations in ZNF114 research include:

• Limited functional characterization: While ZNF114 is predicted to be involved in transcriptional regulation, its specific target genes and regulatory mechanisms remain largely undefined.

• Antibody specificity challenges: As a member of the zinc finger protein family, cross-reactivity with structurally similar proteins poses a challenge for antibody-based studies.

• Lack of structural data: No high-resolution structure of ZNF114 appears to be available, limiting structure-based functional predictions.

• Incomplete interactome mapping: The protein-protein and protein-nucleic acid interaction networks involving ZNF114 are not fully characterized.

Methodological advances to overcome these limitations include:

Recent advances in computational modeling, as demonstrated for zinc receptor proteins like ZnuD , could be applied to predict structural features of ZNF114 and identify potential functional domains, guiding experimental design.