ZNF346 Antibody， Biotin conjugated

What is ZNF346 and what are its key cellular functions?

ZNF346 is a 294 amino acid protein containing four matrin-type zinc fingers that preferentially binds to double-stranded RNA (dsRNA) or RNA/DNA hybrids rather than DNA alone . It functions as a nucleolar zinc finger protein involved in cell growth and survival . ZNF346 plays a critical role in protecting neurons by inhibiting cell cycle re-entry through stimulation of p21 gene expression . Recent research has also revealed its potential involvement in hepatocellular carcinoma (HCC) progression and its association with immune response modulation .

What is the molecular structure of commercially available ZNF346 antibody, biotin conjugated?

The commercially available ZNF346 antibody, biotin conjugated, is typically a polyclonal antibody raised in rabbits against synthetic peptides derived from human ZNF346 . The antibody targets regions within amino acids 181-294 of the human ZNF346 protein . The antibody is conjugated to biotin molecules, which allows for enhanced detection sensitivity through biotin-streptavidin interactions in various immunoassays. The antibody is usually supplied in a storage buffer containing aqueous buffered solution with 0.01M TBS (pH 7.4), 1% BSA, 0.03% Proclin300, and 50% Glycerol .

How does the biotin conjugation enhance the utility of ZNF346 antibody in research applications?

Biotin conjugation provides several methodological advantages in research applications. The strong biotin-streptavidin interaction (one of the strongest non-covalent interactions in nature) significantly amplifies detection sensitivity, which is particularly beneficial when studying proteins expressed at low levels. The biotin tag allows for flexible experimental design through:

• Signal amplification using streptavidin-coupled detection systems

• Multiplexed detection when combined with other detection methods

• Enhanced stability during storage and experimental conditions

• Compatibility with various detection systems including fluorescence, chemiluminescence, and colorimetric methods

• Reduced background in complex tissue samples compared to conventional detection methods

What are the validated applications for ZNF346 antibody, biotin conjugated?

The ZNF346 antibody, biotin conjugated, has been validated for several experimental applications:

• Enzyme-Linked Immunosorbent Assay (ELISA) at dilutions of 1:500-1000

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) at dilutions of 1:200-400

• Immunohistochemistry on frozen sections (IHC-F) at dilutions of 1:100-500

While the unconjugated version has additional validated applications such as immunoblotting at 0.04-0.4 μg/mL concentrations , the biotin-conjugated variant is particularly suited for immunohistochemical applications where signal amplification is beneficial.

How should researchers optimize protocols for using ZNF346 antibody, biotin conjugated in IHC applications?

For optimal results in immunohistochemistry applications with ZNF346 antibody, biotin conjugated, researchers should:

• Tissue preparation: Properly fix tissues with 10% neutral buffered formalin for 24-48 hours before paraffin embedding. For frozen sections, use optimal cutting temperature (OCT) compound and snap-freeze in liquid nitrogen.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes, as ZNF346 epitopes may be masked during fixation.

• Blocking steps: Block endogenous biotin using avidin-biotin blocking kit to prevent non-specific binding, especially in tissues with high endogenous biotin (liver, kidney).

• Antibody dilution optimization: Begin with the manufacturer's recommended dilution range (1:200-400 for IHC-P, 1:100-500 for IHC-F) and optimize through titration experiments.

• Detection system: Use streptavidin-HRP or streptavidin-fluorophore conjugates for visualization, avoiding ABC (avidin-biotin complex) systems which may cause higher background with biotin-conjugated primary antibodies.

• Incubation conditions: Incubate overnight at 4°C to maximize binding while minimizing non-specific interactions.

• Controls: Include both positive controls (tissues known to express ZNF346) and negative controls (omitting primary antibody or using isotype control).

What is the recommended storage and handling procedure to maintain antibody activity?

To maintain optimal activity of the ZNF346 antibody, biotin conjugated:

• Store at -20°C for long-term storage as recommended by manufacturers .

• Avoid repeated freeze-thaw cycles by preparing working aliquots upon receipt.

• When handling, keep the antibody on ice when out of storage.

• Briefly centrifuge the vial before opening to collect solution at the bottom.

• Do not dilute the stock antibody until immediately before use.

• The antibody is typically stable for at least 12 months when stored properly at -20°C .

• Check the storage buffer composition (typically contains glycerol and stabilizing proteins) which helps maintain antibody integrity .

How can ZNF346 antibody be utilized to investigate its role in hepatocellular carcinoma (HCC)?

Recent research has identified ZNF346 as a potential biomarker in HCC with significant prognostic implications . Researchers can leverage the biotin-conjugated ZNF346 antibody to:

What experimental approaches can be used to explore the relationship between ZNF346 and immune cell infiltration in cancer?

To investigate the relationship between ZNF346 and immune cell infiltration in cancer microenvironments:

• Multiplex immunohistochemistry: Use biotin-conjugated ZNF346 antibody in combination with antibodies against immune cell markers. The following table shows key immune cell markers that have demonstrated correlation with ZNF346 expression:

• Flow cytometry analysis: Utilize biotin-conjugated ZNF346 antibody for intracellular staining in combination with immune cell surface markers to quantify correlations between ZNF346 expression and immune cell populations in dissociated tumor samples.

• Single-cell RNA sequencing integration: Correlate ZNF346 expression data from single-cell RNA sequencing with immune cell transcriptional signatures to map relationships at single-cell resolution.

• Spatial transcriptomics: Combine in situ hybridization for ZNF346 with immunohistochemistry using the biotin-conjugated antibody to analyze spatial relationships between ZNF346-expressing cells and immune cell infiltrates.

• Co-culture experiments: Use biotin-conjugated ZNF346 antibody to monitor expression changes in cancer cells co-cultured with various immune cell populations, providing insights into reciprocal regulatory relationships.

How can researchers investigate the molecular mechanisms of ZNF346's RNA-binding properties?

ZNF346 is known to preferentially bind double-stranded RNA or RNA/DNA hybrids rather than DNA alone . To investigate this molecular mechanism:

• RNA immunoprecipitation (RIP): Use biotin-conjugated ZNF346 antibody to immunoprecipitate ZNF346-RNA complexes followed by RNA sequencing to identify specific RNA targets.

• Cross-linking immunoprecipitation (CLIP): Employ UV cross-linking to stabilize ZNF346-RNA interactions before immunoprecipitation with the biotin-conjugated antibody, allowing for precise mapping of binding sites.

• Electrophoretic mobility shift assay (EMSA): Utilize the biotin-conjugated antibody in supershift assays to confirm the specificity of ZNF346-RNA complexes.

• Mutational analysis: Compare wild-type ZNF346 binding to mutants with alterations in zinc finger domains using the biotin-conjugated antibody to detect binding efficiency. Previous research has demonstrated that zinc finger domains are essential for both RNA binding and nucleolar localization .

• Subcellular fractionation: Use the biotin-conjugated antibody to track ZNF346 localization in cellular compartments, particularly focusing on nucleolar enrichment and correlation with RNA distribution.

What are common technical challenges when using ZNF346 antibody, biotin conjugated, and how can they be addressed?

Researchers may encounter several challenges when working with biotin-conjugated ZNF346 antibody:

• High background in IHC applications:

  • Problem: Endogenous biotin in tissues can lead to non-specific staining

  • Solution: Use avidin-biotin blocking kit prior to antibody incubation; consider switching to a biotin-free detection system

• Weak or absent signal:

  • Problem: Insufficient antigen retrieval or antibody concentration

  • Solution: Optimize antigen retrieval methods (try both citrate and EDTA buffers); increase antibody concentration; extend incubation time

• Non-specific binding:

  • Problem: Inadequate blocking or cross-reactivity

  • Solution: Increase blocking time with 5% BSA or 10% normal serum; include 0.1-0.3% Triton X-100 for better penetration

• Inconsistent results between experiments:

  • Problem: Variation in fixation or processing

  • Solution: Standardize fixation protocol; establish positive controls from the same batch of processing

• Discrepancies between RNA and protein expression:

  • Problem: Post-transcriptional regulation of ZNF346

  • Solution: Validate findings using multiple detection methods; consider investigating miRNA-mediated regulation

• Species cross-reactivity issues:

  • Problem: Antibody may react differently across species despite predicted reactivity

  • Solution: Validate antibody in each species of interest; refer to reactivity data (human, mouse, rat, dog, cow, sheep, horse)

What validation experiments should be performed to ensure ZNF346 antibody specificity?

To ensure the specificity of ZNF346 antibody, biotin conjugated, researchers should conduct the following validation experiments:

• Western blot analysis: Though not the primary application for biotin-conjugated antibodies, performing western blot with both conjugated and unconjugated versions can confirm specificity by detecting a single band at the expected molecular weight (~33 kDa for ZNF346).

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to demonstrate signal reduction in the presence of specific blocking peptide.

• Knockdown/knockout controls: Use siRNA or CRISPR to reduce or eliminate ZNF346 expression in cell lines, then verify reduced antibody staining in these models compared to controls. This approach has been validated in HepG2 cells .

• Immunoprecipitation with mass spectrometry: Perform IP with the antibody followed by mass spectrometry to confirm pull-down of ZNF346 and identify potential cross-reacting proteins.

• Multi-antibody comparison: Compare staining patterns with different antibodies targeting different epitopes of ZNF346, such as comparing bs-12235R-Biotin (targeting aa 181-294) with HPA018485 (targeting a different epitope) .

• Species reactivity testing: Validate antibody performance across different species tissues, especially when working with animal models, as reactivity has been predicted for human, mouse, rat, dog, cow, sheep, and horse .

How should researchers interpret ZNF346 expression patterns in different cellular compartments?

ZNF346 demonstrates distinct subcellular localization patterns that can provide insights into its function:

• Nucleolar localization: Strong ZNF346 staining in nucleoli suggests active involvement in RNA metabolism or ribosome biogenesis, as ZNF346's zinc finger domains are required for nucleolar localization .

• Nucleoplasmic distribution: Diffuse nuclear staining outside nucleoli may indicate involvement in transcriptional regulation or RNA processing.

• Cytoplasmic presence: ZNF346 can shuttle between the nucleus and cytoplasm , so cytoplasmic staining may represent active transport or cytoplasmic RNA binding functions.

• Cell type-specific patterns: Variations in expression between different cell types within a tissue may reflect cell-specific functions, particularly in heterogeneous samples like tumor tissues.

When interpreting results, researchers should consider that:

What are the implications of ZNF346 expression correlation with immune cell markers in cancer research?

The strong correlation between ZNF346 expression and immune cell infiltration has significant implications for cancer research:

• Potential as an immunotherapy biomarker: The positive correlation of ZNF346 with immune checkpoint genes suggests it could predict response to immunotherapy .

• Tumor microenvironment modification: Correlations with immunosuppressive cells (Tregs, M2 macrophages, MDSCs) suggest ZNF346 may contribute to an immunosuppressive microenvironment that promotes tumor escape from immune surveillance .

• Inflammation-cancer axis: The association between ZNF346, HBV infection, and inflammatory markers suggests a role in inflammation-driven carcinogenesis .

• Therapeutic targeting considerations: When designing therapies targeting ZNF346, researchers should consider potential effects on immune cell populations and function.

• Prognostic stratification: The correlation between ZNF346 expression and immune cell infiltration provides an opportunity for better prognostic stratification of patients, particularly in HCC where ZNF346 has been associated with poor prognosis .

The table below highlights significant correlations between ZNF346 and key immune regulatory markers:

How can researchers correlate ZNF346 expression data with clinical outcomes in cancer studies?

To effectively correlate ZNF346 expression data with clinical outcomes in cancer studies:

By systematically applying these approaches, researchers can establish the clinical utility of ZNF346 as a prognostic or predictive biomarker in cancer research.