znf687b Antibody

What is ZNF687 and why is it significant in research?

ZNF687 is a C2H2 zinc finger protein that functions as a transcription factor with significant roles in multiple biological processes. Its importance stems from several key findings:

• It was initially identified as a translocation partner gene with RUNX1 in patients with acute myeloid leukemia (AML)

• It shows weak interaction with Ring1/Rnf2 RING finger protein members of the Polycomb group of proteins, suggesting involvement in chromatin-modifying complexes essential for embryonic development and stem cell renewal

• It may participate in a transcriptional network that includes ZNF592 and ZMYMD8

• Mutations in ZNF687 are associated with Paget disease of bone-6

• It has been found markedly upregulated in hepatocellular carcinoma (HCC) and correlates with poor prognosis

Research into ZNF687 is particularly valuable for understanding transcriptional regulation mechanisms and their implications in cancer biology and bone development disorders.

What types of ZNF687 antibodies are available for research applications?

Several types of ZNF687 antibodies have been developed for research purposes:

When selecting an antibody, researchers should consider the specific application needs, target epitope, and validation data available for each product.

How should I validate ZNF687 antibody specificity for my experimental system?

Rigorous validation is critical for ensuring antibody specificity and reliability in ZNF687 research:

• Positive and negative controls: Include cell lines or tissues known to express ZNF687 (such as HCC cell lines) as positive controls . Use tissues with negligible ZNF687 expression (normal liver tissues have been shown to have undetectable levels) as negative controls .

• Knockdown/knockout validation: Perform siRNA knockdown or CRISPR-Cas9 knockout of ZNF687 in your experimental system and confirm reduction in signal with your antibody.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to verify signal specificity.

• Multiple antibody verification: Use antibodies targeting different epitopes of ZNF687 to confirm consistent localization patterns.

• Western blot analysis: Verify a single band of approximately 130 kDa in Western blots from relevant cell lysates .

• Cross-reactivity assessment: If working with non-human models, test for cross-reactivity as most available antibodies are human-specific .

What are the optimal conditions for immunohistochemical detection of ZNF687?

For successful immunohistochemical detection of ZNF687, consider these methodological approaches:

• Fixation: Use 10% neutral buffered formalin fixation for 24-48 hours for optimal epitope preservation.

• Antigen retrieval: Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is typically effective; optimize for your specific antibody.

• Antibody concentration: Start with 5 μg/mL for most anti-ZNF687 antibodies in IHC applications , but titrate to determine optimal concentration for your specific tissue samples.

• Incubation conditions: Overnight incubation at 4°C often yields optimal results for polyclonal antibodies.

• Detection system: HRP-polymer based detection systems often provide better signal-to-noise ratio than ABC methods for ZNF687 detection.

• Counterstaining: Light hematoxylin counterstaining allows better visualization of nuclear ZNF687 expression.

• Scoring: For consistency with published research, consider classifying samples based on percentage of ZNF687-positive cells, where >2% has been associated with clinical significance in HCC samples .

Why might I be getting inconsistent staining patterns with ZNF687 antibodies?

Inconsistent staining patterns can arise from several technical and biological factors:

• Isoform specificity: ZNF687 exists in at least three isoforms, but some antibodies detect only the two largest isoforms . Verify which isoforms are present in your experimental system and which are detected by your antibody.

• Fixation variations: Overfixation can mask epitopes; standardize fixation times and conditions across samples.

• Antibody degradation: ZNF687 antibodies typically have a shelf life of one year; degraded antibodies can yield inconsistent results .

• Expression heterogeneity: ZNF687 expression in tissues can be heterogeneous. In HCC samples, expression ranges from 0-12.6% of cells .

• Cross-reactivity: Some antibodies may cross-react with related zinc finger proteins; validate specificity in your system.

• Technical variables: Inconsistencies in antigen retrieval time, temperature, antibody incubation, or detection method can all contribute to variable staining.

• Sample preparation: Fresh vs. frozen vs. FFPE samples may show different staining patterns due to differential epitope preservation.

What controls should I include when studying ZNF687 expression in cancer samples?

To ensure reliable interpretation of ZNF687 expression in cancer research:

• Normal tissue controls: Include matched adjacent non-tumor tissue; studies have shown ZNF687 is often undetectable in normal liver tissue compared to HCC samples .

• Positive tissue controls: Include samples with known ZNF687 expression (e.g., certain HCC samples).

• Negative antibody controls: Omit primary antibody to assess non-specific binding of secondary detection systems.

• Isotype controls: Include matched isotype control antibodies to assess non-specific binding of primary antibody.

• Cell line controls: Include cell lines with verified high and low ZNF687 expression levels as reference points.

• Gradient expression controls: If possible, include samples with varying expression levels of ZNF687 to establish a dynamic range for your detection system.

• mRNA correlation: Consider parallel qRT-PCR analysis to correlate protein expression with transcript levels.

How can ZNF687 antibodies be used to investigate its role in cancer stem cell maintenance?

Research has implicated ZNF687 in cancer stem cell (CSC) maintenance, particularly in hepatocellular carcinoma . Here are methodological approaches using ZNF687 antibodies:

• CSC marker co-localization: Perform dual immunofluorescence with ZNF687 antibodies and established CSC markers (CD133, EpCAM, CD44) to determine co-expression patterns.

• Sorting-based analysis: Use FACS to isolate CSC subpopulations based on established markers, then analyze ZNF687 expression by western blot or immunofluorescence.

• Spheroid cultures: Compare ZNF687 expression in 2D monolayer versus 3D spheroid cultures (which enrich for stem-like cells) using immunofluorescence.

• ChIP-seq analysis: Use ZNF687 antibodies for chromatin immunoprecipitation followed by sequencing to identify direct gene targets in CSC populations.

• Functional validation: Combine ZNF687 knockdown/overexpression with antibody-based detection of stemness factors (BMI1, NANOG, OCT4) that ZNF687 has been shown to directly regulate .

• Lineage tracing: In appropriate models, combine ZNF687 immunostaining with lineage tracing to follow the fate of ZNF687-expressing cells.

• Patient-derived xenografts (PDX): Analyze ZNF687 expression patterns in PDX models that maintain tumor heterogeneity, focusing on cells with tumor-initiating properties.

What are the methodological considerations when studying ZNF687's interaction with chromatin-modifying complexes?

ZNF687 has been implicated in interactions with chromatin-modifying complexes . Here are methodological approaches to investigate these interactions:

• Co-immunoprecipitation (Co-IP): Use ZNF687 antibodies to pull down protein complexes, followed by western blot analysis of Polycomb group proteins (Ring1/Rnf2) and other chromatin modifiers.

• Proximity ligation assay (PLA): Utilize ZNF687 antibodies in combination with antibodies against suspected interaction partners to visualize protein-protein interactions in situ.

• ChIP-seq and ChIP-reChIP: Perform sequential ChIP with ZNF687 antibodies and antibodies against chromatin modifications (H3K27me3, H3K4me3) or chromatin modifiers to identify co-occupied genomic regions.

• Mass spectrometry analysis: Use ZNF687 antibodies for immunoprecipitation followed by mass spectrometry to identify novel interaction partners.

• FRET/FLIM analysis: For live cell imaging, use fluorescently labeled antibody fragments to study dynamic interactions between ZNF687 and chromatin modifiers.

• Domain-specific antibodies: Utilize antibodies targeting different domains of ZNF687 to determine which regions mediate protein-protein interactions.

• Cellular fractionation: Combine with western blot analysis using ZNF687 antibodies to determine subcellular localization and co-fractionation with chromatin-modifying complexes.

How can ZNF687 antibodies help resolve contradictory findings about its role in different cancer types?

ZNF687 has been implicated in both oncogenic and tumor-suppressive roles depending on the cancer context. Antibody-based approaches to resolve these contradictions include:

• Comprehensive tissue microarray analysis: Use validated ZNF687 antibodies to perform systematic expression analysis across multiple cancer types and correlate with clinical outcomes.

• Subcellular localization studies: Determine if ZNF687 shows different nuclear vs. cytoplasmic localization patterns in different cancer types, which might explain functional differences.

• Isoform-specific detection: Employ antibodies that can distinguish between ZNF687 isoforms to determine if different isoforms predominate in different cancer contexts.

• Post-translational modification analysis: Use phospho-specific or other modification-specific antibodies to determine if ZNF687 undergoes different post-translational modifications in different cancers.

• Context-dependent interactome analysis: Perform immunoprecipitation with ZNF687 antibodies followed by mass spectrometry in different cellular contexts to identify context-specific protein interactions.

• ChIP-seq in multiple cancer types: Compare ZNF687 genomic binding sites across cancer types where it appears to have opposing roles.

• Single-cell analysis: Combine ZNF687 antibodies with single-cell technologies to resolve heterogeneity within tumor samples that might explain apparently contradictory bulk findings.

What methodological approaches can help investigate ZNF687's potential role in transcriptional regulation beyond cancer?

While ZNF687's role in cancer has been studied extensively, its normal physiological functions require further investigation. Here are antibody-based approaches:

• Developmental expression profiling: Use ZNF687 antibodies to track expression patterns during embryonic development, particularly in bone and hematopoietic tissues.

• Cell type-specific expression analysis: Perform co-immunostaining with cell type-specific markers to identify which cell populations normally express ZNF687.

• Stimulus-response studies: Monitor ZNF687 expression and localization changes following various cellular stimuli (growth factors, stress conditions, differentiation signals).

• ChIP-seq in normal tissues: Identify physiological gene targets of ZNF687 in normal tissues to understand its baseline regulatory functions.

• Protein-protein interaction network mapping: Use ZNF687 antibodies for systematic co-IP studies in normal cells to identify physiological interaction partners.

• Transgenic model validation: Validate antibody specificity in transgenic models with modified ZNF687 expression for developmental studies.

• Comparative analysis across species: Use cross-reactive antibodies (when available) to study evolutionary conservation of ZNF687 expression patterns and functions.