ZNF707 Antibody

What is ZNF707 and what biological functions does it serve?

ZNF707 (Zinc Finger Protein 707) is a nuclear protein that may be involved in transcriptional regulation . While its specific function remains under investigation, it belongs to the zinc finger protein family, which typically functions as transcription factors by binding to DNA and regulating gene expression. Understanding ZNF707's role requires experimental validation through techniques such as chromatin immunoprecipitation (ChIP), gene knockdown/knockout studies, and protein-protein interaction analyses to establish its precise function in cellular processes.

What applications are ZNF707 antibodies validated for in research settings?

ZNF707 antibodies have been validated for multiple research applications including Western Blot (WB) with recommended dilutions of 1:500-1:2000, Immunohistochemistry-Paraffin embedded tissues (IHC-P) at dilutions of 1:50-1:200, and Enzyme-Linked Immunosorbent Assay (ELISA) with recommended starting concentrations of 1 μg/mL . These applications enable researchers to detect and quantify ZNF707 protein expression in various experimental systems. Additionally, some antibodies have been validated for Immunofluorescence with recommended dilutions of 0.25-2 μg/mL . Thorough validation includes positive and negative controls to ensure specificity.

What is the immunogen sequence used for generating anti-ZNF707 antibodies?

The immunogen sequence for commercially available anti-ZNF707 antibodies typically corresponds to amino acids 50-180 of the human ZNF707 protein (NP_776192.2) . The full immunogen sequence is: "CSPRPDLVSRLEQWEEPWVE DRERPEFQAVQRGPRPGARK SADPKRPCDHPAWAHKKTHV RRERAREGSSFRKGFRLDTD DGQLPRAAPERTDAKPTAFP CQVLTQRCGRRPGRRERRKQ RAVELSFICGT" . This region was selected for antibody generation based on its antigenicity, accessibility, and uniqueness to ZNF707, reducing the likelihood of cross-reactivity with other zinc finger proteins.

What are the optimal conditions for Western blot detection of ZNF707?

For optimal Western blot detection of ZNF707, researchers should follow these methodological guidelines:

• Sample preparation: Prepare whole cell lysates with complete protease inhibitors

• Loading amount: Load 20-50 μg of total protein per lane

• Gel percentage: Use 10-12% SDS-PAGE gel for optimal separation

• Transfer conditions: Wet transfer at 100V for 1-2 hours or 30V overnight

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute anti-ZNF707 at 1:500-1:2000 or 0.04-0.4 μg/mL

• Incubation: Overnight at 4°C with gentle agitation

• Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5000-1:10000

• Detection: Enhanced chemiluminescence (ECL) system

• Expected band size: ~40-45 kDa

Validation should include appropriate positive controls such as human cell lines known to express ZNF707.

How should researchers optimize immunohistochemistry protocols for ZNF707 detection?

Optimizing immunohistochemistry for ZNF707 detection requires methodical adjustment of several parameters:

Researchers should include both positive controls (tissues known to express ZNF707) and negative controls (primary antibody omission and ideally tissues from knockout models) to ensure specificity and minimize background.

What cross-reactivity considerations should researchers be aware of when using ZNF707 antibodies?

When using ZNF707 antibodies, researchers should consider several potential cross-reactivity issues:

• Other zinc finger proteins: The zinc finger protein family contains numerous members with similar structural domains, particularly in the C2H2 zinc finger regions. Researchers should validate specificity against closely related family members like ZNF703, ZNF704, and ZNF7 .

• Species cross-reactivity: While some antibodies are reactive only with human samples , others show cross-reactivity with mouse and rat ZNF707 . This cross-reactivity should be experimentally validated if working with non-human models.

• Validation approaches:

  • Western blot with recombinant proteins of related zinc finger proteins

  • Immunoprecipitation followed by mass spectrometry

  • Using ZNF707 knockout/knockdown cells as negative controls

  • Peptide competition assays with the immunizing peptide

  • Testing multiple antibodies targeting different epitopes of ZNF707

Thorough validation is essential because non-specific binding could lead to misinterpretation of experimental results, particularly in complex tissues with multiple zinc finger proteins expressed.

How can researchers effectively design ChIP experiments to study ZNF707 binding to DNA?

Designing effective ChIP experiments for ZNF707 requires careful consideration of several methodological aspects:

• Antibody selection: Choose ChIP-grade anti-ZNF707 antibodies validated specifically for ChIP applications. If unavailable, test IP-validated antibodies in ChIP pilot experiments.

• Cross-linking optimization: Since ZNF707 is likely a transcriptional regulator:

  • Test different formaldehyde concentrations (0.5-1.5%)

  • Evaluate cross-linking times (10-20 minutes)

  • Consider dual cross-linking with DSG (disuccinimidyl glutarate) followed by formaldehyde for improved protein-protein interactions

• Sonication parameters:

  • Optimize sonication to achieve DNA fragments of 200-500 bp

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Experimental controls:

  • Input DNA (pre-immunoprecipitation sample)

  • IgG control (non-specific antibody of same isotype)

  • Positive control (antibody against histone mark or known transcription factor)

  • Non-target region primers for qPCR

• Data analysis approaches:

  • ChIP-qPCR for candidate genes based on literature or preliminary experiments

  • ChIP-seq for genome-wide binding analysis

  • Integrate with RNA-seq data to correlate binding with gene expression

This comprehensive approach will help identify genuine ZNF707 binding sites while minimizing false positives and negatives.

What considerations are important when designing co-immunoprecipitation experiments to identify ZNF707 protein interactions?

When designing co-immunoprecipitation (co-IP) experiments to identify ZNF707 interacting partners, researchers should consider:

• Cell lysis conditions:

  • Test different lysis buffers (RIPA vs. NP-40 vs. Digitonin-based)

  • Include appropriate protease and phosphatase inhibitors

  • Optimize salt concentration (150-300 mM NaCl) to preserve interactions while reducing non-specific binding

• Antibody selection:

  • Use antibodies validated for immunoprecipitation applications

  • Consider epitope location to avoid blocking interaction domains

  • Test both N and C-terminal targeting antibodies

• Experimental approach options:

  • Forward IP: Immunoprecipitate ZNF707 and detect interacting partners

  • Reverse IP: Immunoprecipitate suspected partners and detect ZNF707

  • Tandem affinity purification with tagged ZNF707 for higher purity

• Controls:

  • IgG control precipitation

  • Input sample (pre-IP lysate)

  • Reciprocal co-IPs to confirm interactions

  • ZNF707 knockdown/knockout cells as negative controls

• Detection methods:

  • Western blot for candidate interactors

  • Mass spectrometry for unbiased identification of the interactome

  • Proximity ligation assay (PLA) for in situ validation

These considerations will help establish biologically relevant protein-protein interactions while minimizing technical artifacts.

How should researchers approach subcellular localization studies of ZNF707?

For effective subcellular localization studies of ZNF707, researchers should employ a multi-faceted approach:

• Immunofluorescence microscopy:

  • Optimal fixation: 4% paraformaldehyde for 10-15 minutes

  • Permeabilization: 0.1-0.5% Triton X-100 for nuclear proteins

  • Primary antibody: Anti-ZNF707 at 0.25-2 μg/mL

  • Co-staining with organelle markers (DAPI for nucleus, lamin for nuclear envelope)

  • Super-resolution microscopy for detailed subnuclear localization

• Biochemical fractionation:

  • Sequential extraction of cytoplasmic, nuclear soluble, and chromatin-bound fractions

  • Western blot analysis of fractions using anti-ZNF707 antibody

  • Include fraction-specific markers (GAPDH for cytoplasm, Lamin B1 for nuclear envelope, Histone H3 for chromatin)

• Live-cell imaging:

  • GFP-tagged or HaloTag-fused ZNF707 expression

  • Photoactivatable or photoconvertible fluorescent protein fusions for dynamic studies

  • FRAP (Fluorescence Recovery After Photobleaching) to assess mobility

• Electron microscopy:

  • Immunogold labeling for ultrastructural localization

  • Correlative light and electron microscopy for precise localization

• Stimulus response:

  • Examine localization changes upon cell cycle progression, differentiation, or stress conditions

Current evidence suggests ZNF707 is predominantly nuclear , consistent with its proposed function in transcriptional regulation.

How can researchers analyze the potential role of ZNF707 in regulating specific gene expression networks?

To analyze ZNF707's role in gene expression regulation, researchers should implement a comprehensive experimental workflow:

• Genomic binding site identification:

  • Perform ChIP-seq to identify genome-wide binding sites

  • Motif analysis to determine DNA sequence preferences

  • Integration with ATAC-seq or DNase-seq for chromatin accessibility correlation

• Transcriptome analysis:

  • RNA-seq following ZNF707 knockdown/knockout

  • RNA-seq in cells overexpressing wild-type or mutant ZNF707

  • Time-course experiments to distinguish direct from indirect effects

  • Single-cell RNA-seq to identify cell-specific responses

• Epigenetic impact assessment:

  • ChIP-seq for histone modifications (H3K4me3, H3K27ac, H3K27me3)

  • Bisulfite sequencing to analyze DNA methylation patterns

  • FAIRE-seq or MNase-seq for nucleosome positioning

• Functional validation:

  • Luciferase reporter assays with predicted target promoters

  • CRISPR activation/interference at ZNF707 binding sites

  • Site-directed mutagenesis of binding motifs

  • 3C/4C/Hi-C to identify chromatin looping interactions

• Data integration:

  • Network analysis to identify regulated pathways

  • Comparison with publicly available datasets for related zinc finger proteins

  • Multi-omics integration (transcriptome, epigenome, interactome)

This systematic approach will help establish ZNF707's position within regulatory networks and identify directly regulated genes.

What are the most effective approaches for studying ZNF707 post-translational modifications and their functional significance?

To study ZNF707 post-translational modifications (PTMs) and their functional impact, researchers should employ:

• PTM identification:

  • Immunoprecipitation of endogenous ZNF707 followed by mass spectrometry

  • Phospho-specific, acetyl-specific, or ubiquitin-specific antibodies in Western blot

  • 2D gel electrophoresis to separate modified forms

  • Phos-tag gels for phosphorylation detection

• Site-specific analysis:

  • Site-directed mutagenesis of predicted modification sites

  • Generation of phosphomimetic mutants (S/T to E/D) or non-phosphorylatable mutants (S/T to A)

  • CRISPR knock-in of tagged wild-type vs. mutation-bearing ZNF707

• Functional assessment:

  • Chromatin binding (ChIP-seq/ChIP-qPCR) comparison between wild-type and mutant ZNF707

  • Protein-protein interaction changes (co-IP, proximity labeling)

  • Subcellular localization alterations

  • Gene expression effects (RNA-seq, qPCR of target genes)

• Regulatory enzyme identification:

  • Inhibitor screens or genetic screens to identify kinases, phosphatases, acetyltransferases

  • Reverse co-IP with candidate modifying enzymes

  • In vitro modification assays with purified enzymes

• Physiological context:

  • Stimulus-dependent modification changes (growth factors, stress, cell cycle)

  • Cell type-specific modification patterns

  • Disease-associated modification alterations

These approaches will reveal how PTMs regulate ZNF707 function and potentially identify intervention points for modulating its activity.

What methodological approaches should be used to investigate ZNF707 in cancer or other disease contexts?

To investigate ZNF707 in disease contexts, particularly cancer, researchers should implement:

• Expression analysis in clinical samples:

  • Immunohistochemistry on tissue microarrays with quantitative scoring

  • RNA-seq analysis of tumor vs. normal tissue

  • Single-cell analysis to identify cell-specific alterations

  • Correlation with clinical parameters (stage, grade, survival)

• Genomic alterations:

  • Analysis of TCGA and ICGC databases for mutations, copy number variations

  • Whole-exome sequencing to identify novel variants

  • Functional characterization of disease-associated mutations

• Functional studies in disease models:

  • CRISPR knockout/knockdown in disease-relevant cell lines

  • Overexpression of wild-type or mutant ZNF707

  • 3D organoid cultures to recapitulate tissue context

  • Patient-derived xenografts for in vivo studies

• Mechanistic investigations:

  • ChIP-seq in disease vs. normal cells to identify differential binding

  • Interactome analysis in disease context

  • Comparison of regulated genes between normal and disease states

  • Drug response modulation by ZNF707 alterations

• Therapeutic relevance:

  • Correlation with drug sensitivity/resistance

  • Combination approaches with standard therapies

  • Development of assays to monitor ZNF707 activity as biomarker

This systematic approach will help establish whether ZNF707 plays causal roles in disease pathogenesis and evaluate its potential as a diagnostic marker or therapeutic target.

How can researchers troubleshoot non-specific binding issues in Western blot and immunohistochemistry applications?

When encountering non-specific binding with ZNF707 antibodies, researchers should systematically troubleshoot:

• Western blot optimization:

  • Increase blocking stringency (5% BSA or 5% milk in TBST for 1-2 hours)

  • Reduce primary antibody concentration (start with 1:2000 dilution)

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

  • Increase washing duration and frequency (5 × 5 minutes)

  • Test different membrane types (PVDF vs. nitrocellulose)

  • Include 5% normal serum from secondary antibody species

  • Use freshly prepared buffers and reagents

• Immunohistochemistry refinement:

  • Perform antigen retrieval optimization (pH, temperature, duration)

  • Reduce antibody concentration (1:200 dilution)

  • Extend blocking time (2 hours at room temperature)

  • Include avidin/biotin blocking for biotin-based detection systems

  • Add 0.1-0.3% Triton X-100 to reduce background

  • Consider tyramide signal amplification for specific signal enhancement

• Validation and controls:

  • Include blocking peptide competition assays

  • Test antibody on ZNF707-depleted samples

  • Compare results with multiple antibodies targeting different epitopes

  • Include isotype controls at equivalent concentrations

• Sample preparation considerations:

  • Ensure complete protein denaturation for WB

  • Optimize tissue fixation time (overfixation may cause non-specific binding)

  • Use fresh or properly stored samples

These methodical approaches will help distinguish specific ZNF707 signal from background or cross-reactivity with related proteins.

How should researchers validate antibody specificity for ZNF707 in their experimental system?

Comprehensive validation of ZNF707 antibody specificity requires a multi-pronged approach:

• Genetic validation:

  • CRISPR/Cas9 knockout of ZNF707

  • siRNA/shRNA knockdown with multiple targeting sequences

  • Comparison of signal before and after depletion

• Overexpression validation:

  • Transfection with tagged ZNF707 constructs

  • Correlation between tag detection and antibody signal

  • Dose-dependent signal increase with increasing expression

• Peptide competition:

  • Pre-incubation of antibody with immunizing peptide

  • Concentration-dependent signal reduction

  • Non-competing peptide as negative control

• Molecular weight verification:

  • Comparison of observed band with predicted molecular weight

  • Detection of appropriate band shifts with tagged constructs

  • Analysis of isoform-specific detection if applicable

• Cross-reactivity assessment:

  • Testing on closely related zinc finger proteins (ZNF703, ZNF704)

  • Recombinant protein panel testing

  • IP-MS to identify all proteins recognized by the antibody

• Literature correlation:

  • Comparison of results with published data

  • Validation across multiple detection methods (WB, IHC, IF)

What are the best approaches to quantitatively analyze ZNF707 expression levels across different experimental conditions?

For quantitative analysis of ZNF707 expression, researchers should employ:

• Western blot quantification:

  • Include loading controls (β-actin, GAPDH, total protein staining)

  • Run standard curves with recombinant protein

  • Use fluorescent secondary antibodies for broader linear range

  • Employ image analysis software with background subtraction

  • Normalize to multiple housekeeping genes or total protein

• qPCR method:

  • Design primers spanning exon-exon junctions

  • Validate primer efficiency using standard curves

  • Use multiple reference genes validated for stability

  • Apply ΔΔCt or standard curve methods for quantification

  • Include no-RT controls to detect genomic contamination

• Immunohistochemistry quantification:

  • Whole slide scanning for unbiased analysis

  • Automated scoring algorithms (H-score, Allred score)

  • Multiplex IHC to normalize against cell type markers

  • Include calibration standards on each slide

  • Blind scoring by multiple observers for validation

• Flow cytometry:

  • Optimize fixation and permeabilization for nuclear proteins

  • Include fluorescence minus one (FMO) controls

  • Use median fluorescence intensity (MFI) for quantification

  • Include calibration beads for absolute quantification

  • Perform multiparameter analysis for cell type-specific expression

• Proteomics approaches:

  • SILAC or TMT labeling for relative quantification

  • Parallel reaction monitoring (PRM) for targeted quantification

  • Spike-in standards for absolute quantification

  • Multiple peptide quantification for robust results

These methodologies enable accurate quantification while accounting for technical variability and biological heterogeneity.