ZNF282 Antibody, FITC conjugated

What is ZNF282 and why is it important in research?

ZNF282 is a zinc finger protein that functions as a transcription factor with multiple cellular roles. Originally identified as a protein that binds to the U5 repressive element (U5RE) of human T-cell leukemia virus type 1 (HTLV-1), ZNF282 has more recently been characterized as a co-activator for both estrogen receptor α and E2F1 transcription factor . Its importance in research stems from its involvement in breast tumorigenesis and its frequent overexpression in esophageal squamous cell carcinoma (ESCC), where it correlates with poor clinical outcomes . ZNF282 regulates cell cycle progression, apoptosis, migration, and invasion of cancer cells, making it a significant target for cancer research and potential therapeutic development.

What are the available types of ZNF282 antibodies and what are their specific applications?

Multiple types of ZNF282 antibodies are available for research applications, including:

Each antibody has specific applications based on its target region and conjugation, with Western blotting and ELISA being the most common uses .

What is the significance of FITC conjugation in ZNF282 antibodies?

FITC (Fluorescein isothiocyanate) conjugation to ZNF282 antibodies enables direct visualization of the antibody-antigen complex through fluorescence microscopy or flow cytometry without requiring secondary antibodies. This conjugation is particularly valuable for:

• Reducing background signal in multi-color immunofluorescence experiments

• Simplifying staining protocols and reducing experiment time

• Enabling direct detection in flow cytometry applications

• Facilitating co-localization studies with other proteins using differently conjugated antibodies

• Enhancing sensitivity in detecting low-abundance proteins like transcription factors

The FITC-conjugated ZNF282 antibody specifically allows researchers to visualize ZNF282 protein localization within cells and tissues, which is crucial for understanding its nuclear translocation and interaction with other transcription factors like E2F1 .

How can ZNF282 antibodies be used to investigate its role as an E2F1 co-activator in cancer research?

ZNF282 antibodies, including FITC-conjugated versions, can be instrumental in investigating ZNF282's role as an E2F1 co-activator through several sophisticated approaches:

• Co-immunoprecipitation (Co-IP) assays: ZNF282 antibodies can be used to pull down protein complexes containing ZNF282 and E2F1, as demonstrated in TE10 cells where endogenous ZNF282 was shown to bind to E2F1 .

• Chromatin Immunoprecipitation (ChIP) assays: ZNF282 antibodies allow researchers to examine the recruitment of ZNF282 to E2F1 target gene promoters. Research has shown that ZNF282 is directly recruited to the promoters of a subset of E2F1 target genes, including CCNA1 and CDC6, but not to others like the E2F1 gene itself .

• Immunofluorescence co-localization studies: FITC-conjugated ZNF282 antibodies can be used alongside differently labeled E2F1 antibodies to visualize their nuclear co-localization during different cell cycle phases.

• Cell cycle analysis: Flow cytometry using FITC-conjugated ZNF282 antibodies can help correlate ZNF282 expression levels with cell cycle distribution, especially important since knockdown of ZNF282 has been shown to increase G1 phase arrest (87.25% vs 61.34% in control) and decrease S phase (4.88% vs 17.19%) and G2/M phase (7.06% vs 19.92%) proportions .

This approach is particularly valuable for understanding how ZNF282 regulates the expression of specific E2F1 target genes involved in cell cycle progression and tumorigenesis.

What experimental design considerations are important when using ZNF282 antibodies to study its differential expression in normal versus cancer tissues?

When designing experiments to study ZNF282 expression differences between normal and cancer tissues using ZNF282 antibodies, researchers should consider:

• Tissue selection and processing:

  • Include matched normal-tumor pairs from the same patients when possible

  • Consider tissue microarrays for high-throughput analysis

  • Ensure proper fixation to preserve epitope accessibility

• Antibody validation:

  • Validate antibody specificity using positive controls (e.g., cells with known ZNF282 overexpression)

  • Include negative controls (e.g., ZNF282-knockdown tissues/cells)

  • Consider using multiple antibodies targeting different epitopes to confirm findings

• Quantification methods:

  • Develop a standardized scoring system for immunohistochemistry (IHC)

  • Use digital image analysis software for objective quantification

  • Correlate protein expression with mRNA levels when possible

• Statistical analysis:

  • Define appropriate cutoff values for "high" versus "low" expression

  • Correlate expression with clinicopathological parameters and survival

  • Use multivariate analysis to establish ZNF282 as an independent factor

• Follow-up functional studies:

  • Design knockdown/overexpression experiments based on expression data

  • Correlate with cell cycle parameters and apoptosis markers

How can researchers investigate the selective recruitment of ZNF282 to only certain E2F1 target gene promoters?

The selective recruitment of ZNF282 to specific E2F1 target gene promoters represents an intriguing research question that can be addressed through several sophisticated approaches:

• Genome-wide ChIP-sequencing:

  • Perform ChIP-seq with both ZNF282 and E2F1 antibodies

  • Identify genomic regions with overlapping versus distinct binding patterns

  • Analyze the DNA sequence motifs at sites of co-occupancy versus E2F1-only sites

• Sequential ChIP (Re-ChIP) analysis:

  • First immunoprecipitate with E2F1 antibody

  • Then perform a second immunoprecipitation with ZNF282 antibody

  • This identifies genomic regions where both proteins are simultaneously present

• Promoter analysis studies:

  • Create reporter constructs with different E2F1 target promoters

  • Test the effect of ZNF282 overexpression/knockdown on each promoter

  • Mutate potential ZNF282 binding sites to identify critical regions

• Protein domain studies:

  • Generate ZNF282 truncation or point mutation constructs

  • Determine which domains are required for binding to different E2F1 target promoters

  • Correlate with effects on gene expression and functional outcomes

Research has shown that ZNF282 is recruited to the CCNA1 and CDC6 gene promoters but not to the E2F1 gene promoter, consistent with the observation that ZNF282 depletion reduces the expression of CCNA1 and CDC6 but not E2F1 . This suggests that ZNF282 acts as a selective co-activator for only a subset of E2F1 target genes, potentially providing a mechanism for fine-tuning cell cycle regulation.

What are the optimal fixation and antigen retrieval methods when using FITC-conjugated ZNF282 antibodies for immunofluorescence?

When using FITC-conjugated ZNF282 antibodies for immunofluorescence, optimal fixation and antigen retrieval methods are critical for successful detection of this nuclear transcription factor:

• Fixation recommendations:

  • 4% paraformaldehyde (PFA) for 15-20 minutes at room temperature is generally preferred

  • Avoid methanol fixation as it may compromise FITC fluorescence

  • For cultured cells, consider a brief (5 min) permeabilization with 0.1-0.2% Triton X-100 after fixation

  • For tissue sections, freshly frozen tissue followed by acetone fixation (10 min at -20°C) often provides good results

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) at 95-100°C for 20 minutes

  • Allow slow cooling to room temperature to prevent tissue detachment

  • For particularly challenging samples, try sodium EDTA buffer (pH 8.0) as an alternative

• Blocking considerations:

  • Use 5-10% normal serum (from the same species as the secondary antibody would be, if used)

  • Include 0.1-0.3% Triton X-100 in blocking solution for nuclear antigens

  • Consider adding 1% BSA to reduce background

• Special considerations for FITC conjugates:

  • Protect from light during all steps to prevent photobleaching

  • Use anti-fade mounting media containing DAPI for nuclear counterstaining

  • Consider using stronger antigen retrieval for nuclear transcription factors

Since ZNF282 functions as a transcription factor and localizes primarily to the nucleus, ensure proper nuclear permeabilization and consider using confocal microscopy to clearly visualize nuclear versus cytoplasmic distribution.

What controls should be included when validating a new FITC-conjugated ZNF282 antibody for flow cytometry?

Rigorous validation of a FITC-conjugated ZNF282 antibody for flow cytometry requires several essential controls:

• Positive controls:

  • Cell lines with confirmed high ZNF282 expression (e.g., TE9, TE10 ESCC cells)

  • Cells transfected with ZNF282 expression vectors

  • Tissues known to express ZNF282 (processed into single-cell suspensions)

• Negative controls:

  • Isotype control: FITC-conjugated rabbit IgG from the same host species at the same concentration

  • ZNF282 knockdown cells: Cells treated with validated ZNF282 siRNA or shRNA

  • Blocking peptide competition: Pre-incubation of the antibody with the immunizing peptide

• Staining controls:

  • Unstained cells to establish autofluorescence baseline

  • Single-stained controls for each fluorophore when performing multi-color analysis

  • FMO (Fluorescence Minus One) controls when multiple markers are analyzed

• Validation experiments:

  • Titration series to determine optimal antibody concentration

  • Time-course experiments after stimulation expected to alter ZNF282 levels

  • Parallel Western blot to confirm specificity and correlation with flow cytometry results

• Data analysis considerations:

  • Gating strategy documentation

  • Comparison of mean fluorescence intensity (MFI) across experimental conditions

  • Analysis of population shifts rather than just positive/negative designations

Research has shown that ZNF282 expression can be effectively knocked down using shRNA, providing an excellent negative control system for antibody validation . Additionally, ZNF282's role in cell cycle regulation suggests that synchronizing cells at different cell cycle stages might provide varying levels of expression for validation purposes.

How can researchers optimize co-immunoprecipitation protocols when investigating ZNF282 interactions with E2F1 and other transcription factors?

Optimizing co-immunoprecipitation (Co-IP) protocols for investigating ZNF282 interactions with E2F1 and other transcription factors requires several key considerations:

• Lysis buffer optimization:

  • Use a gentle non-denaturing lysis buffer (e.g., 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40)

  • Include protease inhibitors freshly before use

  • For nuclear proteins like ZNF282 and E2F1, consider a specialized nuclear extraction protocol

  • Test different salt concentrations (150-300 mM) to optimize stringency

• Antibody selection and immobilization:

  • For ZNF282 pull-down, select antibodies that target regions not involved in protein-protein interactions

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Cross-link antibodies to beads to prevent co-elution of heavy chains that may interfere with detection

  • Consider using magnetic beads for gentler handling and lower background

• IP conditions:

  • Optimize antibody-to-lysate ratio through titration experiments

  • Perform immunoprecipitation at 4°C overnight with gentle rotation

  • Include appropriate controls: IgG control, input sample (5-10%), and unbound fraction

• Washing conditions:

  • Perform 4-5 washes with ice-cold lysis buffer

  • Consider increasing salt concentration slightly in wash buffer to reduce non-specific interactions

  • For weak interactions, reduce detergent concentration in the final washes

• Detection methods:

  • Western blotting with specific antibodies against ZNF282 and E2F1

  • Consider reverse Co-IP (immunoprecipitate with E2F1 and probe for ZNF282)

  • For comprehensive interaction studies, combine with mass spectrometry analysis

Research has demonstrated successful Co-IP of endogenous ZNF282 and E2F1 in TE10 cells, confirming their physical interaction . Additionally, GST pull-down assays have confirmed direct interaction between ZNF282 and E2F1, providing complementary evidence to Co-IP results .

What are common troubleshooting strategies for weak or non-specific signals when using ZNF282 antibodies?

When encountering weak or non-specific signals with ZNF282 antibodies, researchers can implement the following troubleshooting strategies:

• For weak signals:

  • Increase antibody concentration (perform titration experiments)

  • Extend incubation time (overnight at 4°C)

  • Optimize antigen retrieval methods for immunohistochemistry/immunofluorescence

  • Use signal amplification systems (e.g., biotin-streptavidin)

  • Confirm target protein expression levels in your sample

  • For Western blotting, load more protein or use more sensitive detection reagents

  • For FITC-conjugated antibodies, ensure protection from light and use fresh samples

• For non-specific signals:

  • Increase blocking time and concentration (5-10% normal serum or BSA)

  • Add 0.1-0.3% Tween-20 to washing buffers

  • Perform additional washing steps with larger volumes of buffer

  • Pre-adsorb antibody with cell/tissue lysate from negative control samples

  • Try a different antibody targeting a different epitope of ZNF282

  • For Western blotting, optimize SDS-PAGE conditions and transfer time

• Specificity validation:

  • Perform antibody validation using ZNF282 knockdown samples (confirmed by 74 kDa band reduction)

  • Include positive controls (cell lines with confirmed ZNF282 expression)

  • Consider peptide competition assays to confirm specificity

  • Compare results with different antibodies targeting different epitopes

• Application-specific considerations:

  • For FITC-conjugated antibodies in flow cytometry: optimize permeabilization for nuclear proteins

  • For ChIP applications: optimize cross-linking conditions and sonication parameters

  • For Co-IP: adjust lysis buffer stringency and washing conditions

ZNF282 has been detected as a single strong protein band at approximately 74 kDa by Western blotting, which should serve as a reference point for specificity validation .

How can researchers interpret discrepancies between ZNF282 protein detection and mRNA expression data?

When researchers encounter discrepancies between ZNF282 protein detection (using antibodies) and mRNA expression data, several factors should be considered for proper interpretation:

• Post-transcriptional regulation mechanisms:

  • microRNA-mediated regulation of ZNF282 mRNA

  • RNA-binding protein effects on mRNA stability or translation efficiency

  • Alternative splicing leading to protein isoforms not detected by the antibody used

• Post-translational modifications and protein stability:

  • Ubiquitination and proteasomal degradation affecting protein half-life

  • Phosphorylation states affecting antibody epitope recognition

  • Protein compartmentalization (nuclear localization) affecting extraction efficiency

• Technical considerations:

  • Antibody specificity and sensitivity limitations

  • Different dynamic ranges of protein detection methods versus mRNA quantification

  • Timing discrepancies (mRNA changes often precede protein changes)

  • Sample preparation differences between protein and RNA analyses

• Analytical approach for reconciling discrepancies:

  • Temporal analysis: Track both mRNA and protein over a time course

  • Inhibitor studies: Use translation inhibitors (cycloheximide) or proteasome inhibitors (MG132)

  • Multi-method validation: Compare results from different antibodies and detection methods

  • Subcellular fractionation: Analyze nuclear versus cytoplasmic fractions separately

What considerations are important when analyzing ZNF282 expression in relation to patient survival and clinical outcomes?

When analyzing ZNF282 expression in relation to patient survival and clinical outcomes, researchers should consider several critical factors:

How should researchers design experiments to investigate the selective effect of ZNF282 on specific E2F1 target genes?

To investigate the selective effect of ZNF282 on specific E2F1 target genes, researchers should design comprehensive experiments addressing several key aspects:

• Comprehensive gene expression analysis:

  • Perform RNA-seq or qRT-PCR arrays following ZNF282 knockdown/overexpression

  • Focus on well-established E2F1 target genes (CCND1, CCND2, CCNA1, CCNE1, CDC2, CDC6, CDC25A, etc.)

  • Categorize genes into ZNF282-dependent and ZNF282-independent groups

• Chromatin occupancy studies:

  • Conduct ChIP-seq for both ZNF282 and E2F1 under the same conditions

  • Perform sequential ChIP (Re-ChIP) to identify regions with co-occupancy

  • Compare binding patterns at promoters of genes that are differentially affected by ZNF282

• Promoter activity assays:

  • Clone promoter regions of representative ZNF282-dependent and independent E2F1 target genes

  • Perform luciferase reporter assays under conditions of ZNF282/E2F1 overexpression or knockdown

  • Create promoter deletions to map critical regions for ZNF282 responsiveness

• Protein-protein interaction analysis:

  • Map the domains of ZNF282 that interact with E2F1 using truncation mutants

  • Identify additional cofactors that might influence selectivity using proteomics

  • Perform in vitro DNA binding assays with purified proteins to assess direct effects

• Functional outcomes assessment:

  • Correlate gene-specific effects with cellular phenotypes (cell cycle, apoptosis, migration)

  • Rescue experiments: reintroduce specific E2F1 target genes in ZNF282-depleted cells

  • Use cell cycle synchronization to examine temporal aspects of regulation

Research has shown that ZNF282 depletion significantly inhibited the expression of CCND2, CCNA1, CDC2, and CDC6, but had little effect on CCND1, CCNE1, CDK2, CDC25A, and E2F1 itself . ChIP assays have confirmed that ZNF282 is recruited to the CCNA1 and CDC6 gene promoters but not to the E2F1 gene promoter . These findings provide a foundation for further investigation into the mechanisms underlying this selectivity.

What are the best approaches for studying ZNF282's dual role as both an estrogen receptor co-activator and an E2F1 co-activator?

Investigating ZNF282's dual role as a co-activator for both estrogen receptor α (ERα) and E2F1 requires sophisticated experimental approaches that address potential crosstalk and context-specific functions:

• Temporal and context-dependent analysis:

  • Compare ZNF282 functions in hormone-responsive versus hormone-independent cells

  • Conduct time-course experiments following estrogen stimulation

  • Examine cell-cycle phase-specific interactions with both partners

  • Analyze effects in normal versus cancer cell contexts

• Protein complex characterization:

  • Perform tandem affinity purification coupled with mass spectrometry

  • Investigate whether ZNF282 forms distinct or overlapping complexes with ERα and E2F1

  • Use proximity ligation assays to visualize interactions in situ

  • Map interaction domains using deletion mutants to identify shared versus specific regions

• Genome-wide binding patterns:

  • Conduct ChIP-seq for ZNF282, ERα, and E2F1 under various conditions

  • Identify sites of co-occupancy versus exclusive binding

  • Analyze binding site sequence characteristics and genomic distribution

  • Correlate binding patterns with gene expression changes

• Functional interplay investigations:

  • Examine how estrogen signaling affects ZNF282-E2F1 interactions and vice versa

  • Test whether ZNF282 can simultaneously or sequentially interact with both partners

  • Investigate competition between pathways under limiting ZNF282 conditions

  • Study effects of ZNF282 phosphorylation or other modifications on partner selection

• Disease relevance studies:

  • Compare ZNF282 functions in breast cancer (where ER signaling is prominent) versus ESCC

  • Correlate expression patterns with patient outcomes in different cancer types

  • Investigate potential therapeutic implications of targeting one pathway versus the other

Research has established ZNF282 as both an ERα co-activator important in breast cancer and as an E2F1 co-activator in ESCC where it regulates cell cycle genes . This dual functionality suggests ZNF282 may serve as an integrator of hormone signaling and cell cycle control, potentially explaining its importance in multiple cancer types.

How can FITC-conjugated ZNF282 antibodies be used to study dynamic changes in ZNF282 localization during cell cycle progression?

FITC-conjugated ZNF282 antibodies offer powerful tools for studying dynamic changes in ZNF282 localization during cell cycle progression through several methodological approaches:

• Live-cell imaging techniques:

  • For live-cell applications, consider cell-permeable FITC-conjugated antibody delivery systems

  • Perform time-lapse microscopy to track ZNF282 localization in real-time

  • Co-transfect cells with fluorescently-tagged cell cycle markers (e.g., PCNA-RFP for S phase)

  • Quantify nuclear/cytoplasmic ratios over time as cells progress through the cycle

• Fixed-cell analysis across synchronized populations:

  • Synchronize cells using standard methods (double thymidine block, nocodazole, serum starvation)

  • Collect cells at different time points after release

  • Perform immunofluorescence with FITC-conjugated ZNF282 antibodies

  • Counter-stain with cell cycle markers (cyclin antibodies) and DNA dyes

• Flow cytometry applications:

  • Combine FITC-conjugated ZNF282 antibody staining with DNA content analysis (PI staining)

  • Create bivariate plots of ZNF282 expression versus DNA content

  • Sort cells based on cell cycle phase for subsequent molecular analysis

  • Correlate ZNF282 levels with expression of cell cycle regulators

• High-content imaging analysis:

  • Perform automated microscopy of fixed cells stained with FITC-conjugated ZNF282 antibodies

  • Use image analysis software to quantify nuclear intensity, size, and morphology

  • Correlate with cell cycle phase markers and E2F1 localization

  • Analyze thousands of cells for statistically robust results

• Perturbation studies:

  • Examine ZNF282 localization after treatment with cell cycle inhibitors

  • Compare localization patterns in control versus ZNF282-regulated gene knockdown cells

  • Investigate the effect of E2F1 depletion on ZNF282 localization and vice versa