ZNF268 Antibody

What is ZNF268 and what antibodies are available for its detection?

ZNF268 (zinc finger protein 268) is a transcription factor also known as HZF3. It exists in multiple isoforms, with ZNF268a and ZNF268b2 being the two predominant products of the ZNF268 gene . For detecting ZNF268 in research applications, there are antibodies that recognize either all isoforms or specific variants. The SD antibody can detect total ZNF268 proteins (including both ZNF268a and ZNF268b2), while the E3 antibody specifically recognizes ZNF268a but not ZNF268b2 . These antibodies have been validated in multiple applications including western blotting and immunohistochemistry, making them valuable tools for investigating ZNF268 expression patterns in various tissues and cell lines.

How do I select the appropriate ZNF268 antibody for my specific research application?

• Target specificity: Determine whether you need to detect all ZNF268 isoforms or specific variants

• Application compatibility: Confirm the antibody has been validated for your intended applications (western blot, IHC, IF, etc.)

• Species reactivity: Ensure compatibility with your experimental model

• Validation status: Review literature citations demonstrating the antibody's efficacy in applications similar to yours

For cancer research focusing on isoform expression patterns, using both antibodies in parallel can provide valuable comparative data on the differential expression of ZNF268 isoforms.

What are the validated experimental applications for ZNF268 antibodies?

ZNF268 antibodies have been validated for several experimental applications in research settings. Based on published literature, these applications include:

• Western blotting: ZNF268 antibodies have been successfully used for protein detection in whole cell lysates (WCL), allowing for quantitative assessment of expression levels

• Immunohistochemistry (IHC): Both the SD and E3 antibodies have been employed for tissue staining to visualize ZNF268 expression patterns in normal and cancerous tissues

• Immunoprecipitation: These antibodies can be used to pull down ZNF268 protein complexes for interaction studies

• Chromatin immunoprecipitation (ChIP): For investigating ZNF268 binding to DNA targets in transcriptional regulation studies

When using these antibodies for western blotting, researchers typically prepare whole cell lysates using RIPA buffer followed by centrifugation at 12,000 rpm at 4°C for 10 minutes. Protein concentration determination using the BCA method is recommended before sample preparation for SDS-PAGE .

How should I optimize immunohistochemical staining with ZNF268 antibodies?

Optimizing immunohistochemical staining with ZNF268 antibodies requires careful consideration of several methodological factors:

• Fixation method: Paraffin-embedded tissues have been successfully used with ZNF268 antibodies. Standard formalin fixation and paraffin embedding protocols are suitable

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended to unmask antibody binding sites

• Antibody dilution: Start with manufacturer's recommended dilution (typically 1:100 to 1:500) and optimize based on signal-to-noise ratio

• Incubation conditions: Overnight incubation at 4°C often yields optimal results for primary antibody binding

• Detection system: HRP-conjugated secondary antibodies with DAB substrate have been validated for ZNF268 visualization

• Controls: Always include positive controls (known ZNF268-expressing tissues like certain cervical cancer samples) and negative controls (omitting primary antibody)

Studies examining cervical cancer specimens have successfully utilized both the SD antibody (detecting total ZNF268) and E3 antibody (specific for ZNF268a) to distinguish expression patterns between normal cervical squamous epithelium and cervical carcinoma tissues .

How is ZNF268 expression altered in cervical cancer and what methodologies detect these changes?

Research has demonstrated significant alterations in ZNF268 expression in cervical cancer compared to normal tissue. Specifically, total ZNF268 (detected by the SD antibody) is significantly overexpressed in cervical squamous cell carcinoma specimens compared to normal squamous epithelium (p < 0.001) . Interestingly, expression patterns differ between ZNF268 isoforms—while ZNF268b2 is overexpressed in cancer tissues, ZNF268a shows reduced expression in approximately 40% of carcinomas compared to normal samples .

The following methodological approach has been validated for detecting these expression changes:

• Specimen collection: Paraffin-embedded tissue sections from normal cervical squamous epithelium and cervical squamous carcinoma

• Antibody selection: Use of both SD antibody (detecting total ZNF268) and E3 antibody (specific for ZNF268a)

• Immunohistochemistry: Standard protocols with appropriate antigen retrieval

• Scoring system: Semi-quantitative assessment based on staining intensity and percentage of positive cells

• Statistical analysis: Comparison between normal and cancer tissues using appropriate statistical tests (p-value calculation)

This approach has revealed that ZNF268 overexpression is more significant in cervical cancer than in other cancer types examined, suggesting a potential role in cervical carcinogenesis .

What functional roles of ZNF268 have been demonstrated through antibody-based research approaches?

Antibody-based research approaches have uncovered several functional roles of ZNF268, particularly in cancer development and progression. These findings include:

• Cell proliferation regulation: ZNF268 knockdown in cervical cancer cells causes cell cycle arrest at the G0/G1 phase and reduces colony formation

• Apoptosis modulation: ZNF268 deficiency increases sensitivity to TNFα-induced apoptosis

• Tumor growth promotion: In xenograft models, ZNF268 knockdown suppresses tumor growth with increased apoptosis

• NF-κB signaling enhancement: ZNF268b2 increases NF-κB signaling both in vitro and in vivo

Methodologically, these findings were established through:

• RNA interference: Lentiviral particles expressing shRNA targeting ZNF268 were used to generate knockdown cell lines (common sequences: 5′-CGGGAAAGACTTCAGTAGTAAA-3′ and 5′-GCACGCATGGAAAGAGTTTGAT-3′)

• Western blot analysis: Using validated antibodies to confirm knockdown efficiency and examine downstream signaling effects

• Cell cycle analysis: Flow cytometry with propidium iodide staining

• Apoptosis assays: TNFα treatment combined with Annexin V/PI staining

• Xenograft models: Subcutaneous injection of ZNF268-silenced or control cells in nude mice, followed by tumor measurement and analysis

These methodologies collectively demonstrate that ZNF268, particularly the ZNF268b2 isoform, contributes to cervical carcinogenesis partly through enhancing NF-κB signaling.

How can ChIP assays be optimized when studying ZNF268 interactions with DNA?

Chromatin immunoprecipitation (ChIP) assays provide valuable insights into ZNF268's function as a transcription factor by identifying its DNA binding sites. When optimizing ChIP assays for ZNF268 research, consider the following methodological approach:

• Cross-linking: Treat cells with 1% formaldehyde at room temperature for 15 minutes to cross-link proteins to DNA

• Cell lysis: Wash cells twice with PBS and lyse in SDS lysis buffer

• Chromatin fragmentation: Sonicate lysates on ice to generate DNA fragments of approximately 200-500 bp

• Antibody selection: Use validated antibodies against ZNF268 or its isoforms; additionally, antibodies against known interacting partners (like GATA-1, FOG, CREB-2) may be included for comprehensive analysis

• Immunoprecipitation: Incubate chromatin fragments with antibodies and collect immunoprecipitated complexes using protein A/G-agarose beads

• Reverse cross-linking: Incubate pellets at 65°C for 4 hours followed by proteinase K digestion

• DNA purification and analysis: Purify DNA and analyze by PCR or next-generation sequencing

For negative controls, use IgG antibodies of the same species as your primary antibody. Additionally, include input controls (chromatin samples not subjected to immunoprecipitation) to normalize your results.

What approaches should be used when studying ZNF268 isoform-specific functions in cancer models?

Studying ZNF268 isoform-specific functions in cancer requires sophisticated approaches that can distinguish between the activities of different variants, particularly ZNF268a and ZNF268b2. The following methodological framework is recommended:

• Isoform-specific detection: Use antibodies with validated specificity for different isoforms (E3 for ZNF268a, while total ZNF268 can be detected with SD antibody)

• Expression analysis in clinical samples:

  • Use immunohistochemistry with isoform-specific antibodies

  • Quantify expression levels in normal versus cancer tissues

  • Correlate with clinical parameters and patient outcomes

• Functional studies with isoform selectivity:

  • Design isoform-specific knockdown constructs targeting unique regions

  • Create overexpression models with individual isoforms

  • Assess differential effects on proliferation, apoptosis, and signaling

• Xenograft models:

  • Generate stable cell lines with isoform-specific modulation

  • Inject approximately 1×10^7 cells subcutaneously into nude mice

  • Monitor tumor growth and analyze harvested tumors by real-time PCR and western blot

• Pathway analysis:

  • Examine isoform-specific effects on key signaling pathways like NF-κB

  • Use pathway restoration experiments (e.g., reconstitution of NF-κB activity in ZNF268 knockdown cells)

This comprehensive approach has revealed that ZNF268b2 specifically contributes to carcinogenesis by enhancing NF-κB signaling, while ZNF268a may have distinct functions, as evidenced by its differential expression pattern in cervical cancer tissues.

How should I address non-specific binding when using ZNF268 antibodies?

Non-specific binding is a common challenge when working with ZNF268 antibodies. To address this issue, consider implementing the following methodological approaches:

• Antibody validation:

  • Confirm antibody specificity using positive and negative controls

  • Include ZNF268 knockdown samples as negative controls

  • Use recombinant ZNF268 protein as a positive control

• Blocking optimization:

  • Extend blocking time to 1-2 hours at room temperature

  • Test different blocking agents (5% non-fat milk, 5% BSA, or commercial blocking buffers)

  • Include 0.1-0.3% Tween-20 in wash buffers to reduce background

• Antibody dilution optimization:

  • Perform titration experiments to determine optimal concentration

  • Typical working dilutions range from 1:200 to 1:1000 for western blot

  • For IHC applications, more dilute solutions (1:500 to 1:2000) may be appropriate

• Cross-adsorption:

  • Pre-adsorb antibodies with cell/tissue lysates from ZNF268-negative samples

  • If cross-reactivity with specific proteins is suspected, include recombinant versions of these proteins in pre-adsorption

When interpreting results with potential non-specific binding, always compare patterns across multiple experimental approaches and consider validation through orthogonal methods such as mass spectrometry.

How can I reconcile contradictory data when analyzing ZNF268 expression in different cancer types?

When faced with contradictory data regarding ZNF268 expression across different cancer types, follow this methodological framework for reconciliation:

• Context-specific analysis:

  • Recognize that ZNF268 expression varies significantly between tissue types

  • Tissue microarray analysis has shown that ZNF268 upregulation is most significant in cervical cancer but may show reverse patterns in other tissues

  • Document specific cancer subtypes, stages, and differentiation status

• Isoform-specific considerations:

  • Different ZNF268 isoforms may have opposing expression patterns

  • While ZNF268b2 is overexpressed in cervical cancer, ZNF268a is downregulated in about 40% of cases

  • Always specify which isoform is being detected in your analysis

• Methodological comparison:

  • Create a comparison table documenting different detection methods used across studies

  • Note antibody clones, detection systems, and scoring criteria

  • Evaluate whether differences stem from technical variations rather than biological reality

• Statistical rigor:

  • Ensure adequate sample sizes for statistical power

  • Apply appropriate statistical tests for your data type

  • Consider meta-analysis approaches when integrating multiple datasets

• Functional validation:

  • Complement expression data with functional studies

  • Determine whether observed expression changes translate to functional outcomes

  • Use xenograft models to validate in vitro findings

This systematic approach recognizes that ZNF268's role may be context-dependent, with expression patterns and functions varying across cancer types and even within cancer subtypes.

What emerging technologies can enhance ZNF268 antibody-based research?

Several emerging technologies show promise for advancing ZNF268 antibody-based research:

• Proximity ligation assays (PLA):

  • Enables visualization of protein-protein interactions in situ

  • Can detect ZNF268 interactions with NF-κB pathway components

  • Provides spatial context for molecular interactions within the cell

• Mass cytometry (CyTOF):

  • Allows simultaneous detection of multiple proteins at single-cell resolution

  • Can examine ZNF268 expression alongside numerous signaling markers

  • Enables identification of rare cell populations with unique ZNF268 expression patterns

• Spatial transcriptomics combined with protein detection:

  • Correlates ZNF268 protein localization with gene expression profiles

  • Provides tissue context for understanding ZNF268 function

  • Reveals microenvironmental influences on ZNF268 expression

• CRISPR-based technologies:

  • Enables precise genome editing to study endogenous ZNF268 function

  • Allows tagging of endogenous ZNF268 for visualization and purification

  • Facilitates isoform-specific functional studies through targeted modifications

• Single-molecule imaging:

  • Permits visualization of individual ZNF268 molecules in living cells

  • Enables tracking of ZNF268 dynamics during cellular processes

  • Provides insights into ZNF268 oligomerization and complex formation

These technologies will facilitate more precise characterization of ZNF268's roles in normal physiology and disease pathogenesis, potentially revealing new therapeutic targets.

How can ZNF268 antibodies be employed in studies of resistance mechanisms to cancer therapies?

ZNF268 antibodies can be instrumental in investigating resistance mechanisms to cancer therapies through the following methodological approaches:

• Expression profiling in treatment-resistant models:

  • Compare ZNF268 isoform expression in sensitive versus resistant cell lines

  • Monitor changes in ZNF268 expression during development of resistance

  • Correlate expression patterns with clinical outcomes in patient samples

• Signaling pathway analysis:

  • Use ZNF268 antibodies to investigate its role in therapy-induced signaling changes

  • Focus on NF-κB pathway components given ZNF268's established role in this pathway

  • Examine cross-talk with other resistance-associated pathways

• Combination therapy assessment:

  • Evaluate whether ZNF268 knockdown sensitizes resistant cells to therapy

  • Test combination of ZNF268-targeting approaches with standard treatments

  • Monitor synergistic effects on apoptosis, proliferation, and tumor growth

• Predictive biomarker development:

  • Assess whether ZNF268 expression patterns can predict therapy response

  • Develop standardized immunohistochemical protocols for clinical application

  • Validate in retrospective and prospective patient cohorts

• Mechanistic studies using ZNF268 manipulation:

  • Generate ZNF268 knockdown or overexpression in resistant models

  • Examine effects on sensitivity to TNFα-induced apoptosis as previously demonstrated

  • Investigate changes in cell cycle regulation and colony formation capacity