ZNF169 Antibody

What is ZNF169 and why is it important in research?

ZNF169 is a C2H2-type zinc finger protein with a Kruppel associated box domain (KRAB). It's a 603 amino acid nuclear protein containing thirteen C2H2-type zinc fingers . This transcription factor plays a crucial role in multiple cellular processes, including transcriptional regulation .

Recent studies have shown ZNF169 is significantly upregulated in colorectal cancer tissues compared to normal colon tissue, where it promotes cancer cell growth and proliferation . The gene encoding ZNF169 maps to a region of human chromosome 9q22.3, which has been associated with multiple human diseases including colon cancer, migraine auras, basal cell carcinoma, Gorlin syndrome, and extraskeletal myxoid chondrosarcoma .

What tissue expression patterns are observed for ZNF169?

ZNF169 shows distinctive tissue expression patterns, making it important to understand when designing experiments. It is:

• Highly expressed in kidney

• Weakly expressed in spleen, liver, small intestine, and heart

• Present in the alimentary tract

• Significantly upregulated in colorectal cancer tissues compared to adjacent normal tissues

Understanding these expression patterns is critical when selecting positive and negative control tissues for validating antibody specificity in your experiments.

What applications are ZNF169 antibodies typically used for?

ZNF169 antibodies have been validated for multiple research applications:

When selecting applications, consider the subcellular localization of ZNF169 in the nucleus as a transcription factor .

How do I select the most appropriate ZNF169 antibody for my research?

When selecting a ZNF169 antibody, consider these key factors:

• Target region: Different antibodies target distinct regions of ZNF169 (N-terminal, middle region, C-terminal). For example, some antibodies target amino acids 1-85, 71-120, 131-159, 138-167, or 252-301 . Select based on your experimental needs and protein domains of interest.

• Species reactivity: Most ZNF169 antibodies react with human samples, but some also cross-react with mouse, rat, and other species . Confirm reactivity with your experimental model.

• Applications: Ensure the antibody is validated for your specific application (WB, IHC, FACS, etc.)

• Clonality: Polyclonal antibodies often provide higher sensitivity but potentially lower specificity than monoclonal antibodies. Most available ZNF169 antibodies are rabbit polyclonals .

• Validation data: Review western blot images for expected band size (~68 kDa) and IHC images showing appropriate nuclear localization patterns .

How can I validate the specificity of my ZNF169 antibody?

Antibody validation is crucial for ensuring reliable results. Several approaches are recommended:

• Protein array analysis: Some ZNF169 antibodies have been validated on protein arrays containing 384 different antigens including the target . Consider antibodies with "Supported" specificity scores.

• Western blot validation:

  • Look for the predicted 68 kDa band in positive control tissues (kidney)

  • Compare with negative or low-expression tissues (e.g., heart)

  • Observe decreased signal following siRNA knockdown of ZNF169

  • Test overexpression lysates if available

• IHC validation:

  • Compare staining in colorectal cancer tissues (high expression) with normal colon tissues (lower expression)

  • Verify nuclear localization pattern consistent with transcription factor function

  • Include negative controls (primary antibody omission and isotype controls)

• Independent antibody validation: Compare results using antibodies targeting different epitopes of ZNF169 .

What is the optimal protocol for detecting ZNF169 in colorectal cancer tissues using IHC?

Based on successful protocols from recent studies , follow these methodological steps:

• Tissue preparation:

  • Use 5-μm-thick FFPE tissue sections

  • Include both colorectal cancer tissues and adjacent normal tissues (5 cm from tumor margin) as controls

• Deparaffinization and antigen retrieval:

  • Deparaffinize in xylene and hydrate through graded alcohol series

  • Perform antigen retrieval using citrate buffer (pH 6) at high temperature

  • Block endogenous peroxidase with 3% hydrogen peroxide

• Antibody incubation:

  • Block with 10% goat serum for 1 hour

  • Incubate with ZNF169 antibody at 1:100-1:500 dilution overnight at 4°C

  • For visualization, use a biotinylated secondary antibody (e.g., goat anti-rabbit, 1:2,500 dilution) coupled with streptavidin-HRP

  • Develop with DAB and counterstain with hematoxylin (3-5 minutes)

• Analysis:

  • Examine multiple fields (≥3) at 200× magnification

  • Calculate mean density values as relative expression levels

  • Compare ZNF169 expression between tumor and normal tissues

How can I optimize Western blot conditions for detecting ZNF169?

For optimal ZNF169 detection by Western blot:

• Sample preparation:

  • Use kidney tissue lysates or colorectal cancer cell lines (HCT-116, HT-29, RKO) as positive controls

  • Include both whole cell lysates and nuclear fraction preparations to confirm nuclear localization

  • Ensure adequate protein denaturation with SDS and reducing agents

• Gel selection and transfer:

  • Use 10% SDS-PAGE gels for optimal resolution around 68 kDa

  • Transfer to PVDF membranes with standard protocols

• Antibody conditions:

  • Block with 5% non-fat milk or BSA in TBST

  • Incubate with ZNF169 antibody at 1:1000 dilution overnight at 4°C

  • Use appropriate HRP-conjugated secondary antibody (anti-rabbit IgG at 1:8000-1:10000)

  • Develop using ECL technique

• Expected results:

  • Primary band at 68 kDa (predicted molecular weight)

  • Possible secondary band at ~45 kDa in some samples

  • Stronger signal in colorectal cancer cell lines compared to normal cells

How can I study the role of ZNF169 in gene regulation using ChIP assays?

Chromatin immunoprecipitation (ChIP) can reveal direct targets of ZNF169 as a transcription factor. Based on research protocols demonstrating ZNF169's regulation of ANKZF1 :

• Experimental design:

  • Use colorectal cancer cell lines (HCT-116, HT-29, RKO) with confirmed ZNF169 expression

  • Create control and experimental groups (ZNF169 overexpression and knockdown)

  • Use ZNF169 antibodies confirmed for immunoprecipitation applications

• ChIP protocol optimization:

  • Cross-link cells with 1% formaldehyde (10 minutes)

  • Sonicate chromatin to 200-500 bp fragments

  • Immunoprecipitate with ZNF169 antibody and IgG control

  • Analyze by qPCR using primers for suspected target gene promoters (e.g., ANKZF1 promoter)

• Data analysis:

  • Calculate fold enrichment over IgG control

  • Compare binding between overexpression, knockdown, and control conditions

  • Validate findings with dual luciferase reporter assays to confirm transcriptional activity

• Target confirmation:

  • For confirmed targets like ANKZF1, verify correlation in patient samples

  • Perform rescue experiments by combining ZNF169 modulation with target gene modulation

What are the key considerations when using ZNF169 antibodies for flow cytometry?

When employing ZNF169 antibodies for flow cytometry:

• Sample preparation challenges:

  • Since ZNF169 is a nuclear transcription factor, ensure proper cell permeabilization

  • Use paraformaldehyde fixation (2-4%) followed by permeabilization with 0.1-0.5% Triton X-100 or commercial permeabilization buffers

  • Include nuclear staining to confirm accessibility to nuclear proteins

• Antibody selection:

  • Choose ZNF169 antibodies specifically validated for FACS applications

  • Test antibody concentration ranges (typically starting at 1 μg/ml)

• Controls:

  • Include isotype controls at matched concentrations

  • Use cells with confirmed ZNF169 knockdown as negative controls

  • Include compensation controls if performing multi-color analysis

• Data interpretation:

  • Analyze shifts in fluorescence intensity relative to controls

  • Consider signal in context of cell cycle phases (as transcription factors may show cell-cycle dependent expression)

  • Correlate flow cytometry data with other techniques like Western blot

How can ZNF169 antibodies be used to investigate the ZNF169-ANKZF1 axis in colorectal cancer progression?

Recent research has revealed that ZNF169 regulates ANKZF1 expression in colorectal cancer, with important implications for cancer progression . To investigate this axis:

• Co-expression analysis:

  • Perform dual IHC staining for ZNF169 and ANKZF1 in CRC tissues

  • Calculate correlation coefficients between ZNF169 and ANKZF1 expression levels

  • Compare with patient outcome data to assess prognostic significance

• Mechanism studies:

  • Use ZNF169 antibodies for ChIP-seq to identify genome-wide binding sites

  • Confirm binding to ANKZF1 promoter using ChIP-qPCR

  • Perform dual luciferase reporter assays with wild-type and mutated ANKZF1 promoter constructs

• Functional validation:

  • Design rescue experiments combining ZNF169 overexpression with ANKZF1 knockdown

  • Assess cellular outcomes (proliferation, colony formation, EdU incorporation)

  • Use ZNF169 antibodies to monitor expression in various experimental conditions

• Clinical correlation:

  • Create a scoring system for ZNF169 and ANKZF1 co-expression in patient samples

  • Correlate with clinicopathological features and survival data

  • Identify patient subgroups that might benefit from targeting this axis

How do I resolve contradictory results when detecting ZNF169 with different antibodies?

When facing contradictory results with different ZNF169 antibodies:

• Epitope mapping analysis:

  • Compare the target regions of each antibody (N-terminal, middle region, C-terminal)

  • Consider whether protein modifications or interactions might block specific epitopes

  • Evaluate whether alternative splicing could affect epitope presence

• Validation with genetic approaches:

  • Create ZNF169 knockdown models using siRNAs to confirm signal specificity

  • Generate overexpression systems with tagged ZNF169 to validate antibody detection

  • Consider CRISPR/Cas9 knockout controls for complete validation

• Application-specific troubleshooting:

  • For Western blot discrepancies: Test different lysis methods to ensure complete extraction

  • For IHC/ICC differences: Compare antigen retrieval methods and fixation protocols

  • For flow cytometry variations: Evaluate permeabilization efficiency

• Isoform consideration:

  • Investigate whether different antibodies recognize distinct ZNF169 isoforms

  • The observation of both 68 kDa and 45 kDa bands in Western blots may indicate isoforms or processing

  • Consult RNA-seq data to determine prevalent isoforms in your experimental system

How might ZNF169 antibodies contribute to developing targeted therapies for colorectal cancer?

ZNF169 antibodies could facilitate therapeutic development through:

• Biomarker development:

  • Use validated ZNF169 antibodies in tissue microarrays to identify patient subgroups with high expression

  • Correlate expression with treatment responses to conventional therapies

  • Develop IHC protocols suitable for clinical diagnostic laboratories

• Target validation:

  • Apply antibodies to confirm ZNF169 expression in patient-derived xenografts and organoids

  • Monitor ZNF169 levels following treatment with potential inhibitors

  • Use proximity ligation assays to identify protein interaction partners as alternative targets

• Mechanism exploration:

  • Employ ChIP-seq with ZNF169 antibodies to map the complete regulatory network

  • Identify additional downstream targets beyond ANKZF1

  • Use results to develop therapeutic strategies targeting critical nodes in the network

• Functional antibody approaches:

  • Investigate whether intrabodies against ZNF169 could disrupt its function

  • Study whether antibody-drug conjugates might be developed for targeted delivery

  • Explore ZNF169 internalization mechanisms in cancer cells

What considerations are important when using ZNF169 antibodies for multiplex immunofluorescence studies?

For successful multiplex immunofluorescence with ZNF169 antibodies:

• Panel design:

  • Select antibodies raised in different host species to avoid cross-reactivity

  • When studying the ZNF169-ANKZF1 axis, note that ZNF169 is nuclear while ANKZF1 is cytoplasmic

  • Include markers for cell types of interest (epithelial, immune, stromal)

• Technical optimization:

  • Test antibodies individually before combining into multiplex panels

  • Optimize signal amplification methods for detecting low-abundance transcription factors

  • Consider tyramide signal amplification for increased sensitivity

• Sequential staining approaches:

  • If using same-species antibodies, employ sequential staining with stripping or blocking steps

  • Validate stripping efficiency by confirming removal of primary-secondary complexes

  • Include controls for potential cross-reactivity at each step

• Image analysis considerations:

  • Develop quantitative algorithms to assess nuclear vs. cytoplasmic signals

  • Implement cell segmentation strategies to analyze expression at single-cell level

  • Correlate ZNF169 expression with markers of proliferation and other functional outcomes