ZNF346 Antibody，FITC conjugated

What is ZNF346 and why is it significant in research?

ZNF346, also known as JAZ (Just another zinc finger protein), is a 294 amino acid protein containing four matrin-type zinc fingers that has attracted significant research interest due to its role in transcriptional regulation. The zinc-finger domains within ZNF346 function as DNA-binding modules, allowing the protein to participate in various forms of transcriptional activation or repression within the cell. Matrin-type zinc fingers, which were first identified in the protein matrin-3, share structural similarities with classical C2H2 zinc fingers but possess distinct functional properties that make them particularly interesting to researchers . These specialized domains have been identified in several spliceosome RNA-binding proteins, suggesting ZNF346 may play important roles in RNA processing and gene expression regulation. Due to alternative splicing events, ZNF346 exists in at least two distinct isoforms, adding another layer of complexity to its study and making antibodies against this protein valuable tools for understanding its various functional roles .

What are the primary applications of FITC-conjugated ZNF346 antibodies in research?

FITC-conjugated ZNF346 antibodies serve as powerful tools for visualizing and quantifying ZNF346 protein expression and localization in cells and tissues through various immunological techniques. The primary application of these conjugated antibodies is immunofluorescence (IF) on paraffin-embedded tissues (IHC-P), with recommended dilutions typically ranging from 1:50 to 1:200 depending on specific experimental conditions and tissue types . The direct FITC conjugation eliminates the need for secondary antibody incubation steps, streamlining experimental protocols and reducing potential sources of non-specific binding or background. This direct labeling approach is particularly valuable for co-localization studies where multiple proteins need to be visualized simultaneously, as it allows for more straightforward multiplexing with other fluorophore-conjugated antibodies. Additionally, while not specifically mentioned for the FITC-conjugated variant, ZNF346 antibodies have demonstrated utility in Western blotting applications, as evidenced by validation data showing detection of endogenous ZNF346 in human kidney tissue lysates .

What are the key specifications of FITC-conjugated ZNF346 antibodies?

The FITC-conjugated anti-ZNF346 antibody is a rabbit-derived polyclonal IgG antibody that has been directly labeled with fluorescein isothiocyanate (FITC) fluorophore for direct detection applications. As a polyclonal antibody, it recognizes multiple epitopes on the ZNF346 protein, which can enhance signal strength but may also increase the potential for cross-reactivity compared to monoclonal alternatives . The antibody is developed using rabbit as the host species, which typically provides robust immunogenicity and high-affinity antibody production. While specific concentration information is not provided for the FITC-conjugated variant, related ZNF346 antibodies are typically formulated at concentrations around 700 μg/ml in appropriate buffer systems . The specificity of this antibody has been demonstrated through its ability to detect endogenous levels of total ZNF346 protein in various applications, making it suitable for studying native protein expression without requiring overexpression systems . As with most fluorophore-conjugated antibodies, the FITC conjugation results in an antibody with an absorption maximum around 495 nm and emission maximum around 519 nm, producing the characteristic green fluorescence commonly used in immunofluorescence applications.

What are the recommended storage conditions for maintaining FITC-conjugated ZNF346 antibody activity?

Proper storage of FITC-conjugated ZNF346 antibodies is crucial for maintaining their immunoreactivity and fluorescence properties over time. For long-term storage, FITC-conjugated antibodies should be kept at -20°C, which helps preserve both antibody structure and fluorophore activity . It is important to note that FITC is sensitive to photobleaching, so antibody vials should be protected from light exposure during storage by keeping them in opaque containers or wrapping in aluminum foil. Related ZNF346 antibodies are typically formulated in a stabilizing solution containing pH7.4 PBS with 0.05% sodium azide (NaN3) as a preservative and 40% glycerol to prevent freeze-thaw damage . When working with the antibody, it is advisable to aliquot the stock solution into smaller volumes upon first thawing to minimize repeated freeze-thaw cycles, as each cycle can potentially degrade both the antibody and the conjugated fluorophore. For short-term storage (less than a week), refrigeration at 4°C is generally acceptable, but exposure to room temperature should be minimized. Following these storage recommendations will help ensure reproducible results across experiments and maximize the usable lifetime of the antibody reagent.

What is the recommended protocol for immunofluorescence staining using FITC-conjugated ZNF346 antibodies?

For optimal immunofluorescence staining results with FITC-conjugated anti-ZNF346 antibodies, researchers should follow a carefully optimized protocol that preserves both antigen integrity and fluorophore activity. Begin by fixing tissue sections or cell cultures with an appropriate fixative (typically 4% paraformaldehyde for 10-15 minutes for cultured cells or using formalin-fixed paraffin-embedded tissue sections followed by deparaffinization and rehydration steps). Following fixation, perform antigen retrieval if necessary, which may involve heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) depending on the specific tissue type and fixation conditions . Block non-specific binding sites using a blocking solution containing 5-10% normal serum (from a species different from the antibody host) and 0.1-0.3% Triton X-100 in PBS for 1 hour at room temperature. Incubate samples with the FITC-conjugated anti-ZNF346 antibody at the recommended dilution (1:50-1:200) in antibody diluent overnight at 4°C in a humidified chamber protected from light . After primary antibody incubation, wash samples thoroughly with PBS containing 0.1% Tween-20 (PBST) three times for 5 minutes each. Counterstain nuclei with DAPI (1 μg/ml in PBS) for 5 minutes if desired, followed by additional washing steps. Finally, mount slides using an anti-fade mounting medium to minimize photobleaching during imaging and analysis.

What controls should be incorporated when validating ZNF346 antibody specificity in different experimental contexts?

Implementing rigorous controls is essential for validating the specificity of FITC-conjugated ZNF346 antibodies and ensuring the reliability of experimental results across different applications. Primary negative controls should include the omission of the primary antibody while maintaining all other aspects of the staining protocol, which helps identify background fluorescence from non-specific binding of secondary reagents or autofluorescence of the tissue itself. Peptide competition assays, where the antibody is pre-incubated with excess immunizing peptide or recombinant ZNF346 protein before application to the sample, can demonstrate specificity by showing reduced or abolished staining when the antibody binding sites are blocked . Biological negative controls utilizing tissues or cell lines known not to express ZNF346 should be included to confirm the absence of non-specific binding to unrelated proteins. Positive controls should incorporate samples with confirmed ZNF346 expression, such as human gastric cancer tissue or kidney tissue, which have been previously validated for ZNF346 detection . For advanced validation, researchers should consider using RNA interference (siRNA or shRNA against ZNF346) or CRISPR-Cas9 knockout models to create cells with reduced or absent ZNF346 expression, enabling direct comparison with wild-type cells to confirm antibody specificity. In multi-color immunofluorescence experiments, single-color controls should be included to assess and correct for potential spectral overlap between fluorophores.

How can researchers troubleshoot background fluorescence issues when using FITC-conjugated ZNF346 antibodies?

Excessive background fluorescence is a common challenge in immunofluorescence experiments that can obscure specific signals and complicate data interpretation when working with FITC-conjugated ZNF346 antibodies. Tissue autofluorescence, particularly pronounced in tissues rich in collagen, elastin, or lipofuscin (such as skin, blood vessels, and aging tissues), can be reduced by treating sections with 0.1-1% sodium borohydride in PBS for 10 minutes prior to blocking or by using specialized autofluorescence quenching reagents commercially available for this purpose. Inadequate blocking can lead to non-specific antibody binding, which may be addressed by extending the blocking step duration (up to 2 hours), increasing the concentration of blocking serum (up to 10-15%), or adding 1-5% BSA to the blocking solution to provide additional protein blocking sites. Over-fixation can sometimes create artificial binding sites or increase tissue autofluorescence, so optimizing fixation time and conditions for each tissue type is advisable. If high background persists despite these measures, increasing the number and duration of wash steps (e.g., 5-6 washes of 10 minutes each) with PBST can help remove weakly bound antibodies. Additionally, diluting the FITC-conjugated anti-ZNF346 antibody in blocking solution rather than in antibody diluent alone may provide more effective competition for non-specific binding sites. For tissues with particularly challenging background issues, consider using confocal microscopy with narrower bandpass filters to better discriminate between specific FITC signal and broader autofluorescence spectra.

What are the best approaches for multiplexing FITC-conjugated ZNF346 antibodies with other fluorophore-conjugated antibodies?

Multiplexing fluorescent antibodies enables simultaneous visualization of multiple targets, providing valuable insights into protein co-localization and relative expression patterns within the same sample. When designing multiplexed immunofluorescence experiments incorporating FITC-conjugated ZNF346 antibodies, careful selection of complementary fluorophores with minimal spectral overlap is essential to avoid bleed-through artifacts. Ideal companion fluorophores include those with emission spectra well-separated from FITC (519 nm), such as Cy3 (570 nm), Cy5 (670 nm), or Alexa Fluor 647 (668 nm). When selecting additional antibodies for multiplexing, choose those raised in host species different from the anti-ZNF346 antibody (rabbit) to avoid cross-reactivity issues; antibodies from mouse, goat, or chicken sources are good alternatives. Sequential staining protocols, where each primary and secondary antibody pair is applied and detected before introducing the next set, can help minimize cross-reactivity in complex multiplexing scenarios. When working with tissues known to have high autofluorescence, consider using fluorophores in the far-red spectrum (such as Alexa Fluor 647) for the most important or potentially weakest targets, as tissue autofluorescence is typically lower in this range. Spectral unmixing algorithms available in advanced microscopy software can help separate overlapping fluorescent signals during image analysis when complete spectral separation isn't achievable through filter selection alone. Always include single-stain controls for each fluorophore to establish the spectral profile of each label and facilitate accurate unmixing of the combined signals in multiplexed samples.

What are the emerging applications of ZNF346 antibodies in cancer research?

ZNF346 antibodies are increasingly being utilized in cancer research to investigate the protein's potential roles in tumorigenesis, progression, and treatment response across various malignancies. Immunohistochemical studies using these antibodies have demonstrated differential expression patterns of ZNF346 in cancerous tissues compared to normal counterparts, as evidenced by validation data from human gastric cancer tissue staining . The ability to visualize ZNF346 localization at the cellular and subcellular level using FITC-conjugated antibodies provides researchers with valuable insights into how this zinc finger protein's distribution might change during malignant transformation or in response to therapeutic interventions. ZNF346's potential involvement in transcriptional regulation and RNA binding makes it an interesting candidate for studies investigating dysregulated gene expression programs in cancer cells. Future applications may include high-throughput tissue microarray screening to correlate ZNF346 expression levels with clinical outcomes and treatment responses across large patient cohorts. Combined approaches using both FITC-conjugated antibodies for spatial localization and complementary techniques like chromatin immunoprecipitation (ChIP) could help identify the genomic binding sites and target genes regulated by ZNF346 in specific cancer contexts. As our understanding of zinc finger proteins in cancer biology continues to evolve, ZNF346 antibodies will likely play an increasingly important role in deciphering the molecular mechanisms underlying their contribution to disease pathogenesis and identifying new therapeutic opportunities.