ZFAND3 Antibody

What is ZFAND3 and why is it important in cancer research?

ZFAND3 is an AN1/A20 zinc finger domain containing protein that functions as a transcriptional regulator. It has been identified as a crucial driver of glioblastoma (GBM) invasion through a genome-wide interference screen . ZFAND3 localizes to the nucleus and binds to promoter regions to regulate the expression of several genes related to cancer cell invasion and adhesion, including COL6A2, FN1, and NRCAM 3 . Its importance in cancer research stems from its role in regulating invasive properties of GBM cells - knockdown of ZFAND3 significantly reduces invasion capacity, while overexpression can confer invasive properties to previously non-invasive cells . These findings suggest ZFAND3 may serve as a potential therapeutic target for reducing cancer invasiveness.

What types of ZFAND3 antibodies are commercially available for research?

Several types of ZFAND3 antibodies are available for research applications:

The rabbit polyclonal antibody from Affinity Biosciences detects endogenous levels of ZFAND3 in human, mouse, and rat samples . The mouse monoclonal antibody from Bio SB is specifically validated for immunohistochemistry applications and is available in both concentrate and predilute formats3 . Both antibodies have been demonstrated to be effective for detection of ZFAND3 protein in research contexts.

What are the main applications for ZFAND3 antibodies in experimental research?

ZFAND3 antibodies have been validated for several key research applications:

• Immunohistochemistry (IHC): Detection of ZFAND3 in paraffin-embedded tissue sections, particularly in glioblastoma samples where nuclear localization correlates with invasive phenotypes3 .

• Western Blot (WB): Detection of ZFAND3 protein (approximately 25kD) in denatured protein samples, useful for confirming knockdown or overexpression efficiency .

• Immunofluorescence (IF): Visualization of ZFAND3 subcellular localization, which is critical for understanding its function as ZFAND3 activity requires nuclear localization .

• ELISA: Detection of ZFAND3 in protein samples with high sensitivity .

• Chromatin Immunoprecipitation (ChIP): Though not explicitly mentioned in the search results, ChIP has been used to demonstrate ZFAND3 association with promoter regions of invasion-related genes .

The choice of application depends on your specific research question, with IHC being particularly useful for examining expression in patient samples and IF/ChIP for mechanistic studies of ZFAND3 function.

How can I use ZFAND3 antibodies to study its role in transcriptional regulation?

ZFAND3 functions as a transcriptional activator through binding to promoter regions of invasion-related genes. To study this role:

• Chromatin Immunoprecipitation (ChIP): Use FLAG-tagged ZFAND3 and corresponding antibodies to perform ChIP-qPCR experiments. Research has shown ZFAND3 associates with zinc finger consensus sites in the promoter regions of COL6A2, NRCAM, and FN1 . Design primers that target these predicted zinc finger binding sites.

• Luciferase Reporter Assays: Clone promoter sequences of genes of interest (e.g., COL6A2, NRCAM, FN1) into luciferase reporter constructs. Co-express these with ZFAND3 or ZFAND3 mutants lacking zinc finger domains to assess transcriptional activation capacity .

• Co-immunoprecipitation: Use ZFAND3 antibodies to precipitate protein complexes involved in transcriptional regulation. Research indicates ZFAND3 acts within a nuclear protein complex to activate gene transcription .

• Immunofluorescence Co-localization: Combine ZFAND3 antibodies with antibodies against known transcriptional regulators to visualize their co-localization at nuclear sites.

The mechanistic data suggests ZFAND3 induces expression of invasion-related genes through activation of a transcriptional complex involving PUF60, which ultimately enhances invasive behavior of GBM cells .

How should I design experiments to investigate ZFAND3's role in cancer cell invasion?

When investigating ZFAND3's role in cancer cell invasion, a multi-faceted experimental approach is recommended:

• Knockdown and Overexpression Studies: Use shRNA for knockdown of ZFAND3 in invasive cell lines and overexpression vectors in non-invasive lines. Confirm these manipulations at both RNA and protein levels using qPCR and western blotting with ZFAND3 antibodies .

• In Vitro Invasion Assays: Employ Boyden chamber assays to quantify invasion capacity following ZFAND3 manipulation. Studies show that ZFAND3 knockdown significantly reduces invasion compared to controls in highly invasive glioblastoma stem cells .

• Ex Vivo Brain Slice Models: Implant modified cells into ex vivo brain slices to better mimic the brain microenvironment. Quantify:

  • Area of colonization

  • Cellular velocity through single-cell tracking
    These parameters were significantly reduced upon ZFAND3 knockdown and increased with overexpression .

• In Vivo Intracranial Models: Perform intracranial implantation of cells with modified ZFAND3 expression and quantify invasion to the contralateral hemisphere. Research has demonstrated fewer invading cells in ZFAND3 knockdown tumors compared to controls .

• Domain Mutation Analysis: Generate constructs with mutations in zinc finger domains or nuclear localization sequences to assess their functional importance in invasion, as both nuclear localization and intact zinc finger domains are required for ZFAND3 activity .

This comprehensive approach will provide robust evidence regarding ZFAND3's role in cancer invasion from molecular mechanisms to in vivo relevance.

What are the optimal protocols for ZFAND3 immunohistochemistry in brain tumor samples?

For optimal ZFAND3 immunohistochemistry in brain tumor samples, follow these methodological guidelines:

• Tissue Preparation:

  • Use formalin-fixed paraffin-embedded (FFPE) tissue sections (4-6μm thickness)

  • Include positive controls (glioblastoma samples with known ZFAND3 expression) and negative controls (omitting primary antibody)

• Antigen Retrieval:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Heating for 20 minutes at 95-98°C is typically effective

• Antibody Selection and Dilution:

  • For mouse monoclonal antibody (Bio SB BSB-169): Use predilute format or dilute concentrate 1:100-1:2003

  • For rabbit polyclonal antibody: Optimal dilution should be determined empirically, starting at 1:200

• Detection System:

  • Use a polymer-based detection system compatible with the host species of primary antibody

  • DAB (3,3'-diaminobenzidine) produces a brown precipitate that effectively contrasts with hematoxylin counterstain

• Evaluation Parameters:

  • Assess both nuclear and cytoplasmic staining patterns, as ZFAND3 function requires nuclear localization

  • Quantify the percentage of positive cells and staining intensity

  • Pay particular attention to invasive regions of the tumor

• Interpretation:

  • Strong nuclear ZFAND3 expression correlates with invasive phenotype in glioblastoma3

  • Compare expression patterns between tumor core and invasive front

This protocol has been validated in multiple studies examining ZFAND3 expression in glioblastoma tissues and provides reliable results for both diagnostic and research applications.

How can I validate the specificity of a ZFAND3 antibody?

To ensure the specificity of a ZFAND3 antibody for your research applications, implement the following validation strategies:

• Western Blot Analysis:

  • Verify a single band at the expected molecular weight (~25kD for ZFAND3)

  • Include positive controls (cell lines with confirmed ZFAND3 expression)

  • Include negative controls (ZFAND3 knockdown samples)

• Knockdown/Overexpression Controls:

  • Compare antibody staining in wild-type cells versus those with shRNA-mediated ZFAND3 knockdown

  • Examine staining in cells overexpressing ZFAND3 versus control cells

  • Signal intensity should correlate with expression levels

• Peptide Competition Assays:

  • Pre-incubate the antibody with the immunizing peptide before application

  • This should block specific binding and eliminate true ZFAND3 signal

• Multiple Antibody Comparison:

  • Test multiple antibodies raised against different epitopes of ZFAND3

  • Consistent staining patterns with different antibodies increases confidence in specificity

• Immunoprecipitation-Mass Spectrometry:

  • Perform immunoprecipitation with the ZFAND3 antibody

  • Confirm the presence of ZFAND3 in the precipitated material using mass spectrometry

• Immunofluorescence Pattern Analysis:

  • ZFAND3 should show both cytoplasmic and nuclear localization, with stronger nuclear signal in invasive cells 3

  • In knockdown cells, staining should be reduced, with particularly notable reduction in nuclear staining

These validation steps are essential before using a ZFAND3 antibody for critical experiments, especially when investigating novel aspects of ZFAND3 biology or developing it as a potential diagnostic marker.

What is known about ZFAND3's mechanism of action in promoting cancer cell invasion?

ZFAND3 promotes cancer cell invasion through several interconnected molecular mechanisms:

• Transcriptional Activation of Invasion-Related Genes: ZFAND3 binds to and activates the promoters of key invasion-related genes including:

  • COL6A2 (collagen type VI alpha 2 chain)

  • FN1 (fibronectin 1)

  • NRCAM (neuronal cell adhesion molecule)
    These genes encode extracellular matrix and adhesion proteins that facilitate cell migration and invasion.

• Nuclear Protein Complex Formation: ZFAND3 functions within a nuclear protein complex to activate gene transcription. Research indicates it interacts with transcriptional regulators including PUF60 .

• Zinc Finger Domain Dependency: ZFAND3's activity requires intact zinc finger domains. Mutations in these domains abolish its ability to activate target gene promoters in luciferase reporter assays .

• Nuclear Localization Requirement: ZFAND3 must localize to the nucleus to exert its pro-invasive functions. In glioblastoma cells, ZFAND3 displays both cytoplasmic and nuclear localization, with nuclear accumulation correlating with invasive capacity3 .

• Sequence-Specific DNA Binding: Analysis of promoter regions of ZFAND3 target genes revealed GC-rich target sequences preferentially recognized by zinc finger containing DNA-binding proteins. ChIP-qPCR experiments confirmed ZFAND3 association with these predicted zinc finger consensus sites .

This transcription factor-like activity of ZFAND3 creates a gene expression program that ultimately enhances the invasive behavior of cancer cells, particularly in glioblastoma. The mechanism appears conserved, as ZFAND3 overexpression can confer invasive properties to previously non-invasive cells .

How does ZFAND3 expression correlate with clinical outcomes in glioblastoma patients?

While the search results don't provide direct data on clinical outcomes correlation, the functional data suggests important clinical implications:

• Invasion and Surgical Resection: The infiltrative nature of glioblastoma, partially driven by ZFAND3, critically prevents complete surgical resection and masks tumor cells behind the blood-brain barrier, reducing the efficacy of systemic treatment .

• Therapeutic Resistance: Invasive cells that migrate away from the tumor core escape surgical resection and are partially sheltered from radio- and chemotherapy .

• Detection Limitations: Invasive cells are often not detected by standard imaging techniques, complicating patient monitoring .

• Multicellular Network Formation: Glial tumors form multicellular networks through ultra-long membrane protrusions (tumor microtubes) that facilitate brain invasion and contribute to treatment resistance .

• Biomarker Potential: The nuclear localization of ZFAND3 in invasive glioblastoma cells suggests its potential utility as a biomarker for identifying aggressive, invasive tumors3.

Further research investigating direct correlations between ZFAND3 expression levels, localization patterns, and patient outcomes (survival, recurrence, treatment response) would be valuable to fully understand its clinical significance. The established functional role of ZFAND3 in promoting invasion warrants such clinical correlation studies, as it represents a potential therapeutic target for reducing GBM invasiveness and improving treatment outcomes.

What are common problems encountered when using ZFAND3 antibodies and how can they be resolved?

When working with ZFAND3 antibodies, researchers may encounter several common issues:

• Weak or Absent Nuclear Staining:

  • Problem: Nuclear localization is critical for ZFAND3 function, but may be difficult to detect.

  • Solution: Optimize antigen retrieval conditions; try EDTA buffer (pH 9.0) for enhanced nuclear antigen retrieval. Ensure adequate permeabilization in IF protocols. Use fresh tissue samples or properly fixed specimens to preserve nuclear antigens3 .

• High Background Staining:

  • Problem: Non-specific binding can obscure true ZFAND3 signal.

  • Solution: Increase blocking time (2-3 hours at room temperature); use a combination of BSA, normal serum from the secondary antibody species, and 0.1-0.3% Triton X-100. Optimize antibody dilution and incubation conditions .

• Discrepancies Between Protein and mRNA Levels:

  • Problem: ZFAND3 protein expression may not correlate with mRNA levels.

  • Solution: Validate findings using multiple detection methods (IHC, WB, IF). Consider post-transcriptional regulation mechanisms when interpreting results .

• Inconsistent Staining Patterns Between Different Antibodies:

  • Problem: Different antibodies may give different staining patterns.

  • Solution: Validate with functional assays (e.g., using samples with known ZFAND3 knockdown or overexpression). Compare monoclonal (Bio SB) and polyclonal (Affinity Biosciences) antibodies to confirm patterns 3.

• Difficulties in Co-IP Experiments:

  • Problem: ZFAND3 interactions with transcriptional complexes may be difficult to preserve.

  • Solution: Use gentle lysis conditions; consider crosslinking prior to lysis; optimize salt concentrations in washing buffers to preserve protein-protein interactions .

• Storage and Stability Issues:

  • Problem: Antibody efficacy may decrease over time.

  • Solution: Store antibodies according to manufacturer recommendations (typically at -20°C in 50% glycerol). Avoid repeated freeze-thaw cycles by preparing small aliquots .

When troubleshooting, document all experimental conditions systematically and change only one variable at a time to identify the optimal protocol for your specific application.

What controls should be included when using ZFAND3 antibodies in experimental research?

To ensure reliable and interpretable results when using ZFAND3 antibodies, incorporate these essential controls:

1. Positive Controls:

• GBM tissue sections with known ZFAND3 expression3

• Cell lines with confirmed high ZFAND3 expression (e.g., invasive GBM cell lines)

• ZFAND3-overexpressing cells created through transfection/transduction

2. Negative Controls:

• Primary antibody omission control to assess secondary antibody specificity

• ZFAND3 knockdown cells (shRNA or CRISPR)

• Non-expressing or low-expressing tissues/cells

• Isotype control antibodies matching the primary antibody class and species

3. Specificity Controls:

• Peptide competition assay (pre-incubation of antibody with immunizing peptide)

• Comparison of staining patterns with multiple antibodies targeting different ZFAND3 epitopes

4. Technical Controls:

• Housekeeping proteins (for western blot loading control)

• Nuclear marker (e.g., DAPI) to verify nuclear localization in IF/IHC

• Endogenous peroxidase blocking validation for IHC

5. Experimental Design Controls:

• For knockdown studies: non-targeting shRNA control

• For overexpression studies: empty vector control

• For functional assays: wild-type cells alongside experimental manipulations

6. Application-Specific Controls:

• For ChIP experiments: IgG control, input control, and positive control regions (known ZFAND3 binding sites)

• For luciferase assays: promoterless vector and mutated binding site controls

Incorporating these controls is essential for generating robust, reproducible, and interpretable data when studying ZFAND3 in cancer research contexts.