ZNF346 Antibody, HRP conjugated

What is ZNF346 and what structural features make it unique among zinc finger proteins?

ZNF346, also known as JAZ (Just Another Zinc finger protein), is a 294 amino acid protein containing four matrin-type zinc fingers. Unlike conventional zinc finger proteins, ZNF346 represents a distinct subfamily of RNA-binding Cys2His2 (C2H2) zinc finger proteins. The matrin-type zinc finger structure was first identified in the protein matrin-3 and shows similarities to classical DNA-binding C2H2 zinc fingers . These matrin-type zinc fingers have been identified in several spliceosome RNA-binding proteins, suggesting a specialized role in RNA processing. Two isoforms of ZNF346 exist due to alternative splicing events, which may have implications for its functional diversity .

What are the optimal storage conditions for maintaining ZNF346 antibody activity?

For optimal antibody performance and stability, store ZNF346 antibody at -20°C for long-term preservation (up to one year). For frequent use and short-term storage, the antibody can be kept at 4°C for up to one month . It is critical to avoid repeated freeze-thaw cycles as they can lead to protein denaturation and loss of antibody activity. The antibody is typically supplied in a stabilizing solution containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.2, which helps maintain its structural integrity during storage . When handling the antibody, use sterile technique and minimize exposure to room temperature to preserve antibody performance.

What are the recommended dilutions for ZNF346 antibody in different experimental applications?

These dilution ranges serve as initial guidelines, but researchers should perform titration experiments with appropriate positive and negative controls to determine the optimal antibody concentration for their specific experimental conditions, including cell/tissue type and detection system .

What controls should be included when using ZNF346 antibody in experimental protocols?

A robust experimental design for ZNF346 antibody applications should include multiple controls to ensure result validity:

• Positive control: Use cell lines or tissues known to express ZNF346 (e.g., HepG2 cells have been validated) .

• Negative control: Include samples where ZNF346 is absent or has been knocked down through siRNA techniques .

• Isotype control: Use non-specific rabbit IgG at the same concentration as the ZNF346 antibody to identify non-specific binding.

• Secondary antibody-only control: Omit the primary antibody to assess background from the secondary detection system.

• Blocking peptide competition: Pre-incubate the antibody with the immunizing peptide to verify signal specificity .

• Cross-reactivity assessment: When studying multiple species, validate species-specific reactivity as the antibody is reported to recognize human, mouse, and rat ZNF346 .

These controls help distinguish specific ZNF346 signal from background or non-specific interactions, particularly important when optimizing new experimental protocols or working with complex tissue samples.

How can researchers effectively validate ZNF346 knockdown experiments?

Validation of ZNF346 knockdown requires a multi-level approach to confirm reduced expression at both RNA and protein levels:

• RT-qPCR analysis: Measure ZNF346 mRNA levels following siRNA transfection to confirm knockdown efficiency at the transcriptional level. This method has been successfully applied in studies with HepG2 cells .

• Western blot verification: Analyze protein levels using the ZNF346 antibody (HRP conjugated) at dilutions of 1:500-1:2000 to confirm reduced expression at the protein level .

• Functional assays: Implement cell viability (e.g., CCK-8 assay) and migration assays (wound healing and transwell assays) to assess the functional consequences of ZNF346 knockdown .

• Rescue experiments: Re-introduce ZNF346 expression in knockdown cells to confirm phenotype specificity.

• Dose-response assessment: Use multiple siRNA concentrations to establish a correlation between knockdown efficiency and phenotypic changes.

Research has demonstrated that effective knockdown of ZNF346 in HepG2 cells significantly reduces cell viability and inhibits migration capacity, confirming its role in promoting cell proliferation and migration in hepatocellular carcinoma models .

What is the association between ZNF346 and cancer prognosis, particularly in hepatocellular carcinoma?

ZNF346 has emerged as a significant prognostic biomarker in hepatocellular carcinoma (HCC), with several key clinical correlations:

These findings suggest that ZNF346 detection and quantification using specific antibodies may have clinical utility for risk stratification and treatment planning in HCC patients, particularly those with HBV infection.

How does ZNF346 influence the tumor immune microenvironment and what are the implications for immunotherapy?

ZNF346 plays a complex role in shaping the tumor immune microenvironment with significant implications for immunotherapy response:

• Immune cell correlation: ZNF346, according to the Tumor Immune Estimation Resource (TIMER) database, shows significant positive correlation with infiltrating immune cells in HCC, including B cells, CD8 T cells, CD4 T cells, macrophages, dendritic cells, and neutrophils .

• Immunosuppressive cell association: Strong positive correlations exist between ZNF346 expression and immunosuppressive cells including:

  • M2 macrophages

  • Regulatory T cells (Tregs)

  • Myeloid-derived suppressor cells (MDSCs)

  • Cancer-associated fibroblasts (CAFs)

• Immunosuppressive marker correlation: ZNF346 expression correlates with specific markers of immunosuppressive cells as shown in this data table:

• Inflammatory response: HBV infection may lead to ZNF346 overexpression, which is accompanied by elevated apoptosis and chronic inflammation .

• Immunotherapy implications: ZNF346 expression is associated with poor immunotherapy efficacy, potentially due to its role in promoting an immunosuppressive microenvironment .

When designing studies examining ZNF346's role in the tumor microenvironment, researchers should consider multiplexed approaches combining ZNF346 antibody staining with immune cell markers to analyze spatial relationships between ZNF346-expressing cells and the surrounding immune infiltrate.

What molecular mechanisms underlie ZNF346's role in cell cycle regulation and apoptosis?

The molecular mechanisms of ZNF346 involve complex interactions with key cellular pathways:

• p53 interaction: ZNF346 directly interacts with p53 protein and has a regulatory effect on its function. This interaction influences downstream genes involved in cell cycle arrest and apoptosis .

• Functional domains: The matrin-type zinc fingers in ZNF346 likely contribute to its RNA-binding capabilities, suggesting a role in post-transcriptional regulation .

• Apoptotic regulation: HBV infection leading to ZNF346 overexpression is associated with elevated apoptosis, suggesting a role in cell death pathways .

• Cell proliferation: Experimental knockdown of ZNF346 in HepG2 cells dramatically reduces cell viability, indicating its pro-proliferative function. This was quantitatively demonstrated using CCK-8 assays (p < 0.01) .

• Migration capacity: ZNF346 positively regulates cell migration, as shown through wound healing and transwell assays following ZNF346 knockdown (p < 0.01) .

• Subfamily characteristics: ZNF346 represents a unique subfamily of zinc finger proteins distinct from conventional C2H2 ZNFs, suggesting specialized molecular functions .

When investigating these mechanisms, researchers should consider combining ZNF346 antibody detection with co-immunoprecipitation studies to identify interaction partners, and chromatin immunoprecipitation followed by sequencing (ChIP-seq) to determine DNA binding sites if applicable.

What strategies can address non-specific binding when using ZNF346 antibody in complex tissue samples?

Non-specific binding is a common challenge when working with antibodies in complex tissue samples. For ZNF346 antibody applications, consider these methodological approaches:

• Optimize blocking conditions: Use 3-5% BSA or 5-10% normal serum from the same species as the secondary antibody to reduce non-specific binding sites.

• Titrate antibody concentration: Start with recommended dilutions (1:100-500 for IHC-P, 1:100-1000 for WB) but perform systematic titration to find the optimal signal-to-noise ratio for your specific sample type .

• Adjust incubation parameters: Reduce incubation temperature (4°C overnight instead of room temperature) and extend washing steps to eliminate weak non-specific interactions.

• Use peptide competition: Pre-incubate the antibody with increasing concentrations of the immunizing peptide to confirm signal specificity and identify non-specific binding .

• Employ antigen retrieval optimization: Test multiple antigen retrieval methods (heat-induced vs. enzymatic) and buffer compositions to improve specific epitope accessibility.

• Consider tissue-specific autofluorescence: In fluorescence-based detection, use appropriate quenching methods for tissues with high autofluorescence (particularly liver tissue, which is relevant for HCC studies).

• Implement dual staining validation: Use antibodies against established ZNF346-interacting proteins (such as p53) to confirm co-localization patterns .

These strategies help distinguish true ZNF346 signal from background or non-specific binding, particularly important when analyzing heterogeneous tumor samples with variable expression levels.

How can researchers distinguish between the different isoforms of ZNF346 using antibody-based detection methods?

Distinguishing between ZNF346 isoforms requires careful consideration of antibody epitope location and complementary techniques:

• Epitope mapping: Determine whether the ZNF346 antibody recognizes an epitope common to all isoforms or is isoform-specific. Contact the manufacturer for detailed epitope information if not provided .

• Western blot resolution: Use high-percentage (10-12%) polyacrylamide gels with extended run times to achieve better separation of closely sized isoforms. The calculated molecular weight of ZNF346 is approximately 32.9 kDa, but isoforms may show subtle size differences .

• 2D gel electrophoresis: Combine isoelectric focusing with SDS-PAGE to separate isoforms that may have similar molecular weights but different isoelectric points.

• Isoform-specific knockdown: Design siRNAs targeting unique regions of specific isoforms and confirm differential detection with the antibody .

• Mass spectrometry validation: Following immunoprecipitation with ZNF346 antibody, use mass spectrometry to identify peptides unique to different isoforms.

• RT-PCR complementation: Design primers spanning exon junctions specific to each isoform and correlate mRNA expression with protein detection patterns.

• Recombinant isoform standards: Express recombinant versions of each ZNF346 isoform to serve as size and reactivity standards in Western blot applications.

These approaches allow researchers to determine which ZNF346 isoforms are being detected in their experimental system, crucial for accurately interpreting functional studies given that alternative splicing may generate proteins with distinct biological activities.

What emerging applications of ZNF346 antibody show promise for biomarker development in cancer diagnostics and prognostics?

Several emerging applications of ZNF346 antibody show significant promise for clinical biomarker development:

• Diagnostic screening: With an area under the curve (AUC) of 0.946 in TCGA cohort analysis, ZNF346 detection shows strong potential as a diagnostic biomarker for HCC .

• Prognostic stratification: ZNF346 expression correlates with survival outcomes in HCC, suggesting utility in patient risk stratification and treatment planning .

• Therapeutic response prediction: The association between ZNF346 and immunosuppressive microenvironments suggests potential utility in predicting immunotherapy response .

• Multiplexed tissue analysis: Combining ZNF346 antibody with other markers in multiplexed immunohistochemistry panels could improve tumor classification and identify patient subgroups.

• Liquid biopsy development: Explore detection of ZNF346 in circulating tumor cells or extracellular vesicles as a non-invasive biomarker approach.

• Imaging applications: Developing radio-labeled or fluorescently tagged ZNF346 antibodies for in vivo imaging could enable visualization of ZNF346-expressing tumors.

• Therapeutic targeting validation: ZNF346 antibodies could help validate the efficacy of emerging therapeutic approaches targeting this protein or its downstream pathways.

Each of these applications requires rigorous validation across diverse patient cohorts and careful correlation with clinical outcomes before clinical implementation.

How might ZNF346 contribute to therapeutic resistance mechanisms, and what methodological approaches can investigate this phenomenon?

Investigating ZNF346's potential role in therapeutic resistance requires sophisticated experimental approaches:

• Resistance model development: Establish cell line models with acquired resistance to standard therapies (chemotherapy, targeted therapy, immunotherapy) and assess ZNF346 expression changes using the antibody at optimal dilutions (1:100-1000) .

• Patient-derived xenografts: Compare ZNF346 expression in treatment-naïve and post-treatment residual tumors using standardized immunohistochemistry protocols.

• Single-cell analysis: Implement single-cell RNA sequencing and protein analysis to identify ZNF346-expressing subpopulations that persist after treatment.

• Combination therapy assessment: Evaluate whether ZNF346 knockdown sensitizes cells to standard therapies through viability assays (CCK-8) and apoptosis measurements .

• Pathway interaction mapping: Investigate ZNF346 interactions with known resistance mechanisms, particularly p53 pathway alterations, through co-immunoprecipitation and functional studies .

• In vivo modulation: Develop inducible ZNF346 knockdown systems in mouse models to test the impact on treatment response in vivo.

• Clinical correlation studies: Analyze pre- and post-treatment patient samples to determine whether ZNF346 expression changes correlate with treatment outcomes and recurrence patterns.

These approaches can help determine whether ZNF346 represents a potential therapeutic target to overcome resistance in cancers where it is overexpressed, particularly HCC associated with HBV infection .