ZNF207 Antibody, HRP conjugated

What is ZNF207 and what are its biological functions?

ZNF207 (Zinc finger protein 207) serves multiple critical cellular functions with significant implications for both developmental biology and disease states. At the molecular level, ZNF207 functions as a transcription factor that binds to specific DNA sequences through its zinc finger domain . In embryonic stem cells, ZNF207 forms protein complexes with master pluripotency factors including OCT4 and SOX2, contributing to maintenance of the pluripotent state . Notably, ZNF207 also functions as a kinetochore- and microtubule-binding protein (also known as BugZ) that plays essential roles in spindle assembly during cell division . Through phase transition mechanisms, ZNF207/BugZ forms temperature-dependent liquid droplets that promote microtubule bundling, concentrate tubulin, and enhance microtubule polymerization . Additionally, ZNF207 regulates mitotic chromosome alignment by mediating the stability and kinetochore loading of BUB3, although the exact mechanisms remain under investigation .

What are the recommended storage and handling protocols for ZNF207 Antibody, HRP conjugated?

ZNF207 Antibody, HRP conjugated should be stored at -20°C or -80°C immediately upon receipt . The antibody is typically supplied in liquid form containing preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol in 0.01M PBS, pH 7.4) . For optimal performance and longevity, it is critical to avoid repeated freeze-thaw cycles as these can significantly decrease antibody activity . When aliquoting for long-term storage, prepare single-use volumes in sterile microcentrifuge tubes to minimize freeze-thaw damage. Prior to use, thaw the antibody on ice and briefly centrifuge to collect the solution at the bottom of the tube. For reconstitution of lyophilized antibody, add 50 μL of distilled water to achieve a final concentration of 1 mg/mL . Working dilutions should be prepared fresh on the day of the experiment and kept on ice while in use.

What are the optimal dilution and application protocols for Western blot detection of ZNF207?

For Western blot applications, ZNF207 antibody is recommended to be used at a concentration of 1 μg/mL (approximately 1:1000 dilution from a 1 mg/mL stock) . The detection system should include HRP-conjugated secondary antibody diluted at 1:50,000 - 1:100,000 . For optimal results, prepare protein samples in RIPA buffer supplemented with protease inhibitors. Load 20-40 μg of total protein per lane on 8-12% SDS-PAGE gels, as ZNF207 has a molecular weight of approximately 60-70 kDa. After transfer to PVDF or nitrocellulose membrane, block with 5% non-fat dry milk in TBST for 1 hour at room temperature. Incubate with diluted primary ZNF207 antibody overnight at 4°C with gentle agitation. Following 3-5 washes with TBST (5 minutes each), incubate with HRP-conjugated secondary antibody for 1 hour at room temperature. After final washes, develop using ECL detection reagents. The specificity of the antibody has been validated across human, mouse, and rat samples .

How can ZNF207 antibodies be utilized in stem cell research applications?

In stem cell research, ZNF207 antibodies provide valuable tools for investigating pluripotency mechanisms and cellular reprogramming processes. For immunofluorescence detection in human embryonic stem cells (hESCs), dilute the primary antibody at 1:100-1:500 in blocking buffer and co-stain with pluripotency markers such as OCT4, SOX2, and NANOG to assess correlation patterns . For ChIP experiments to identify ZNF207 binding sites on chromatin, use 5 μg of antibody per immunoprecipitation reaction with chromatin prepared from 1-2 million cells . When studying ZNF207's role in reprogramming, the antibody can be used to track protein expression dynamics during the conversion of somatic cells to induced pluripotent stem cells (iPSCs). In these applications, Western blot analysis at sequential time points post-transduction with reprogramming factors can reveal how ZNF207 expression changes correlate with acquisition of pluripotency . For studying naïve versus primed pluripotent states, comparative immunostaining between different culture conditions provides insights into ZNF207's differential roles in these distinct pluripotent states .

How does ZNF207 interact with the core pluripotency network, and what are the best methods to detect these interactions?

ZNF207 forms molecular complexes with core pluripotency factors OCT4 and SOX2 through protein-protein interactions, as demonstrated by co-immunoprecipitation studies in hESCs . ChIP-Seq analysis has revealed that ZNF207 binding sites frequently co-localize with OCT4/SOX2 across the genome, with the OCT4/SOX2 consensus motif being the most significantly enriched motif in ZNF207-bound regions (E-value of 5.3E−059) . To effectively investigate these interactions, researchers should employ multiple complementary approaches:

• Co-immunoprecipitation (Co-IP): Lyse cells in non-denaturing buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1% NP-40) supplemented with protease inhibitors. Pre-clear lysates with protein A/G beads before immunoprecipitation with 2-5 μg anti-ZNF207, anti-OCT4, or anti-SOX2 antibodies overnight at 4°C. After washing, analyze by Western blot to detect interacting partners.

• Proximity Ligation Assay (PLA): For in situ detection of protein interactions, use Duolink® PLA technology with primary antibodies against ZNF207 and OCT4/SOX2, followed by oligonucleotide-linked secondary antibodies that generate fluorescent signals when proteins are in close proximity (<40 nm).

• ChIP-reChIP: To identify genomic regions co-bound by ZNF207 and OCT4/SOX2, perform sequential chromatin immunoprecipitation using antibodies against ZNF207 followed by OCT4 or SOX2.

• RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins): Combine ChIP with mass spectrometry to identify the complete protein complex associated with ZNF207 on chromatin.

The functional significance of these interactions can be assessed by generating ZNF207 mutants lacking specific protein interaction domains and testing their ability to rescue pluripotency in ZNF207-depleted cells .

What experimental strategies are most effective for studying ZNF207's role in mitotic spindle assembly?

ZNF207 (also known as BugZ) contributes to spindle assembly through its ability to undergo phase transition, forming liquid droplets that promote microtubule bundling and tubulin concentration . To effectively study this function:

• Live-cell imaging with fluorescently tagged ZNF207: Generate stable cell lines expressing ZNF207-GFP fusion proteins to visualize its dynamic localization during mitosis. Use time-lapse confocal microscopy with z-stacks to track ZNF207 condensates in relation to spindle formation.

• Phase separation assays: Purify recombinant ZNF207 protein and perform in vitro phase separation experiments under varying conditions (temperature, salt concentration, protein concentration) to characterize the biophysical properties of ZNF207 condensates.

• Microtubule co-sedimentation assays: Mix purified ZNF207 with polymerized microtubules, centrifuge to pellet microtubules and associated proteins, then analyze supernatant and pellet fractions by SDS-PAGE to quantify ZNF207's microtubule-binding capacity.

• Domain mapping experiments: Generate truncated or point-mutated versions of ZNF207 to identify regions required for liquid droplet formation and microtubule bundling. Express these constructs in ZNF207-depleted cells to assess functional rescue of spindle assembly defects.

• Structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM): Apply super-resolution imaging techniques to visualize the nanoscale organization of ZNF207 in relation to spindle microtubules and kinetochores.

For all approaches, comparing wild-type ZNF207 with the DNA-binding domain deletion mutant (ZNF207/MUT) provides critical insights into the separation of its transcriptional versus structural functions in mitosis .

How can researchers effectively investigate ZNF207's role in cancer immunology, particularly in hepatocellular carcinoma (HCC)?

Recent findings suggest ZNF207 may function as an immunosuppressive factor in HCC, correlating with poor prognosis and altered immune cell infiltration patterns . To investigate this role:

• Multiplex immunohistochemistry/immunofluorescence: Perform co-staining of ZNF207 with immune cell markers (CD8, CD4, FOXP3, CD11b, CD68) in HCC tissue microarrays to analyze spatial relationships between ZNF207 expression and immune infiltrates. This technique allows visualization of up to 8 markers simultaneously on a single tissue section.

• Single-cell RNA sequencing: Apply scRNA-seq to dissociated HCC tumors with varying ZNF207 expression levels to characterize cell-type-specific effects on the tumor microenvironment at high resolution.

• CRISPR-mediated ZNF207 knockout in HCC models: Generate ZNF207-deficient HCC cell lines and assess their growth in immunocompetent versus immunodeficient mouse models to determine immune-dependent effects.

• T-cell co-culture assays: Culture ZNF207-expressing or ZNF207-depleted HCC cells with activated CD8+ T cells and measure T-cell proliferation, cytokine production, and cytotoxicity to assess functional immunosuppression.

• Cancer-immunity cycle analysis: Apply the framework developed in search result to systematically analyze how ZNF207 affects each step of the cancer-immunity cycle, from antigen release to T-cell killing of cancer cells.

• Combination therapy testing: Evaluate anti-PD1 therapy in ZNF207-high versus ZNF207-low HCC models to determine whether ZNF207 status affects immunotherapy response, as suggested by preliminary findings .

Data analysis should include correlation studies between ZNF207 expression and established immune signatures, particularly focusing on cytotoxic T-cell function markers (CD8A) and complement immune responses (C2, C3) .

What are the optimal ChIP protocols for studying ZNF207 binding to target genes, and how should data be analyzed?

To effectively perform ChIP experiments for ZNF207 targeting:

Sample Preparation and Chromatin Immunoprecipitation:

• Crosslink 10-20 million cells with 1% formaldehyde for 10 minutes at room temperature, then quench with 0.125 M glycine.

• Lyse cells and sonicate chromatin to generate fragments of 200-500 bp (optimize sonication conditions for your cell type).

• Immunoprecipitate using 5 μg of ZNF207 antibody per reaction, with IgG as a negative control.

• Include input controls (non-immunoprecipitated chromatin) at approximately 5% of starting material.

• After washes, reverse crosslinks, treat with proteinase K, and purify DNA using phenol-chloroform extraction or commercial kits.

Analysis Approaches:

• ChIP-qPCR: Design primers targeting the transcription start site (TSS) regions of candidate genes identified in RNA-Seq after ZNF207 knockdown . Include primers for known binding sites as positive controls and gene desert regions as negative controls.

• ChIP-Seq: Prepare libraries from immunoprecipitated DNA following standard protocols and sequence to a depth of at least 20 million uniquely mapped reads. Analyze data using:

  • Peak calling software (MACS2) to identify ZNF207 binding sites

  • Motif enrichment analysis (HOMER or MEME) to identify consensus binding sequences

  • Genome browser visualization to examine binding patterns

  • Correlation with gene expression data to identify direct regulatory targets

• Integrated Analysis: Combine ChIP-Seq with RNA-Seq data to define genes that are both bound by ZNF207 and differentially expressed upon ZNF207 knockdown, as demonstrated in the study that identified 177 directly upregulated and 357 directly downregulated ZNF207 target genes .

For validation of direct targets, perform reporter assays using luciferase constructs containing ZNF207 binding regions. Site-directed mutagenesis of the binding motif should abolish ZNF207-dependent regulation if the binding is functionally significant .

What methods are most effective for studying ZNF207's role in cellular reprogramming, and what controls should be included?

ZNF207 significantly impacts reprogramming efficiency, with knockdown reducing efficiency approximately tenfold and overexpression enhancing it threefold . To effectively study this process:

Experimental Design:

• Reprogramming System Setup:

  • Use established reprogramming protocols (e.g., Yamanaka factors OSKM delivery via retrovirus, Sendai virus, or episomal vectors) in human fibroblasts

  • Modulate ZNF207 levels through siRNA knockdown, shRNA stable knockdown, or overexpression of wild-type or mutant ZNF207

  • Include proper controls: non-targeting siRNA/shRNA, empty vector overexpression

• Comprehensive Assessment Methods:

  • Colony counting after alkaline phosphatase (AP) staining at standardized timepoints (days 14, 21, 28)

  • Flow cytometry for early reprogramming markers (SSEA-4, TRA-1-60) at days 7-14

  • Single-cell tracking to distinguish enhanced reprogramming from proliferative effects

  • Time-course RNA-Seq to identify stage-specific effects of ZNF207

• Functional Validation:

  • Pick and expand multiple iPSC clones from each condition

  • Perform pluripotency assays (embryoid body formation, teratoma assays)

  • Assess differentiation capacity into all three germ layers

Critical Controls:

• Monitor cell proliferation rates in all conditions to distinguish true reprogramming effects from proliferative advantages (as noted in the research, ZNF207 knockdown reduced proliferation to approximately 75% of control)

• Include domain-specific ZNF207 mutants (particularly DNA-binding domain deletion) to dissect mechanism, as mutated ZNF207 failed to promote reprogramming

• Measure endogenous OCT4 activation levels as a direct readout of ZNF207's effect on key pluripotency networks

• Compare effects in different somatic cell types (fibroblasts, blood cells, epithelial cells) to assess cell-type specificity

Analysis should include quantification of reprogramming kinetics (time to colony emergence), efficiency (percentage of successfully reprogrammed cells), and quality (expression levels of pluripotency factors in resulting iPSCs) .

What are common troubleshooting strategies for optimizing ZNF207 antibody performance in different applications?

When working with ZNF207 antibody, researchers may encounter several technical challenges. Below are methodological solutions for common issues:

For Western Blotting:

• Weak or No Signal:

  • Increase primary antibody concentration (try 2-5 μg/mL)

  • Extend primary antibody incubation time to overnight at 4°C

  • Use more sensitive detection methods (e.g., enhanced chemiluminescence plus)

  • Ensure sample contains adequate ZNF207 protein (use positive control lysates)

  • Check transfer efficiency with reversible staining (Ponceau S)

• High Background:

  • Increase blocking time (2-3 hours) or concentration (5-10% milk/BSA)

  • Use different blocking agent (switch between milk and BSA)

  • Increase wash duration and number (5 washes for 10 minutes each)

  • Decrease secondary antibody concentration (try 1:100,000 dilution)

For Immunofluorescence/Immunohistochemistry:

• Optimize Antigen Retrieval:

  • Compare heat-induced (citrate buffer pH 6.0, EDTA pH 8.0) and enzymatic methods

  • Adjust retrieval time (10-30 minutes)

• Signal Enhancement:

  • Implement tyramide signal amplification (TSA) for low-abundance targets

  • Use biotin-streptavidin amplification systems

  • Apply Sudan Black B (0.1% in 70% ethanol) to reduce autofluorescence

For ChIP Applications:

• Low IP Efficiency:

  • Increase antibody amount (5-10 μg per reaction)

  • Extend incubation time (overnight plus 2-4 hours)

  • Pre-clear lysates more thoroughly

  • Optimize chromatin fragmentation (check fragment size on agarose gel)

• Cross-Validation Approaches:

  • Use multiple ZNF207 antibodies targeting different epitopes

  • Perform IP-Western blot before ChIP to confirm antibody efficiency

  • Include spike-in controls for normalization

These optimization strategies should be systematically implemented with appropriate controls to identify the optimal conditions for your specific experimental system .

What experimental design considerations are important when comparing ZNF207 function across different cell types and disease models?

When investigating ZNF207 across diverse cellular contexts and disease models, several methodological considerations are critical for generating reliable, comparable data:

Standardization and Controls:

• Expression Baseline Establishment:

  • Quantify endogenous ZNF207 levels (protein and mRNA) across all cell types using identical methods

  • Create a standardized expression index normalized to housekeeping genes/proteins

  • Generate calibration curves with recombinant ZNF207 for absolute quantification

• Knockdown/Overexpression Consistency:

  • Use identical vectors and regulatory elements across cell types

  • Verify equivalent knockdown/overexpression efficiency (aim for >90% reduction or 3-5 fold increase)

  • Employ inducible systems (e.g., Tet-On/Off) to achieve comparable expression timing

Functional Readouts:

• Context-Appropriate Assays:

  • Stem cells: alkaline phosphatase staining, pluripotency marker expression

  • Cancer cells: proliferation, invasion, immunological markers (CD8, CD4)

  • Mitotic cells: spindle assembly, chromosome alignment, BUB3 stability

• Multi-dimensional Analysis:

  • Combine bulk approaches with single-cell techniques

  • Integrate transcriptomic, proteomic, and functional data

  • Perform time-course experiments rather than single endpoints

Disease Model Considerations:

• Model Selection Strategy:

  • Match models to specific research questions (e.g., immunocompetent models for HCC immune studies)

  • Include both in vitro (cell lines, organoids) and in vivo (animal models) systems

  • Consider patient-derived xenografts for higher clinical relevance

• Cross-Species Validation:

  • Confirm antibody cross-reactivity in different species (human, mouse, rat)

  • Account for species-specific differences in ZNF207 sequence and function

  • Validate key findings across multiple species when possible

Data Analysis Framework:

• Implement multivariate analysis to account for cell type-specific factors

• Use hierarchical clustering to identify cell types with similar ZNF207 functions

• Develop computational models that predict context-dependent functions

By systematically addressing these considerations, researchers can distinguish universal versus context-specific functions of ZNF207 across different biological systems .

How can researchers accurately quantify and interpret ZNF207 expression data in correlation with disease progression?

Accurate quantification and interpretation of ZNF207 expression in disease contexts, particularly cancer progression, requires systematic methodological approaches:

Quantification Methods:

• RNA Expression Analysis:

  • qRT-PCR with validated reference genes specific to tissue type

  • Digital droplet PCR for absolute quantification

  • RNA-Seq with appropriate normalization (TPM/FPKM)

  • RNA in situ hybridization for spatial context

• Protein Quantification:

  • Western blot with densitometry (referenced to loading controls)

  • ELISA using calibrated standards (1:312500 dilution recommended)

  • Quantitative immunohistochemistry with digital image analysis

  • Reverse phase protein arrays for high-throughput screening

Data Collection Frameworks:

Interpretation Strategies:

• Establish Normal Baseline:

  • Compare with matched non-diseased tissue

  • Account for tissue-specific expression patterns

  • Consider cell type composition differences

• Context Integration:

  • Correlate with established cancer immunity cycle scores

  • Analyze relationship with known prognostic factors

  • Perform pathway enrichment analysis of co-expressed genes

• Validation in Independent Cohorts:

  • Use multiple patient cohorts

  • Employ meta-analysis techniques

  • Validate findings across different technological platforms

• Causality Assessment:

  • Determine if ZNF207 expression changes are causes or consequences

  • Establish experimental models to test causality

  • Use conditional probability and Bayesian networks

For HCC specifically, researchers should stratify patients based on ZNF207 expression levels and systematically analyze correlations with immune markers (CD8A, CD4, FOXP3, CD11b, CD68, C2, C3) to determine immunological significance, as demonstrated in previous research .

What are the most promising approaches for targeting ZNF207 in combination with immunotherapy for cancer treatment?

Based on recent findings implicating ZNF207 in immunosuppression in HCC, several strategic approaches for combination therapy with immune checkpoint inhibitors show promise:

Therapeutic Targeting Strategies:

• Small Molecule Inhibitors:

  • Target the DNA-binding domain of ZNF207 to disrupt transcriptional activity

  • Design inhibitors that disrupt protein-protein interactions between ZNF207 and its cofactors

  • Develop degraders (PROTACs) that specifically target ZNF207 for proteasomal degradation

• Genetic Approaches:

  • Use siRNA/shRNA encapsulated in tumor-targeting nanoparticles

  • Apply CRISPR/Cas9 strategies for in vivo gene editing

  • Develop antisense oligonucleotides targeting ZNF207 mRNA

• Combinatorial Treatment Designs:

  • Sequential therapy: ZNF207 inhibition followed by anti-PD1 treatment

  • Concurrent administration with optimized dosing schedules

  • Triple combinations including anti-CTLA4 or other checkpoint inhibitors

Experimental Design for Evaluation:

• Preclinical Models:

  • Orthotopic HCC models in immunocompetent mice

  • Patient-derived xenografts in humanized mice

  • Ex vivo tumor slice cultures treated with combinations

• Biomarker Development:

  • ZNF207 expression as predictive biomarker for immunotherapy response

  • Monitoring changes in T-cell infiltration (CD8A) following treatment

  • Multiplex immunohistochemistry panels including ZNF207 and immune markers

• Resistance Mechanisms Identification:

  • Analyze adaptive responses to ZNF207 targeting

  • Identify compensatory pathways activated upon ZNF207 inhibition

  • Develop strategies to overcome acquired resistance

The most promising approach based on current evidence is the combination of ZNF207 inhibition with anti-PD1 therapy, as research suggests ZNF207 suppresses the HCC microenvironment and may limit immunotherapy efficacy . This combination could potentially convert "cold" tumors (lacking T-cell infiltration) to "hot" tumors that respond to checkpoint inhibition by addressing the negative correlation between ZNF207 expression and cytotoxic T-cell infiltration .

How might advances in single-cell technologies enhance our understanding of ZNF207's role in development and disease?

Single-cell technologies offer unprecedented opportunities to dissect ZNF207 functions with cellular resolution, revealing heterogeneity that bulk analyses cannot detect:

Single-Cell Methodologies and Applications:

• scRNA-Seq for Expression Profiling:

  • Map ZNF207 expression across developmental trajectories

  • Identify rare cell populations with unique ZNF207 dependencies

  • Correlate ZNF207 with lineage-specific transcription factors

  • Application example: Analyzing preimplantation embryos to confirm ZNF207 activation at the 8-cell stage and correlation with NANOG expression

• Single-Cell ATAC-Seq for Chromatin Accessibility:

  • Identify cell type-specific regulatory elements associated with ZNF207

  • Map chromatin state changes following ZNF207 perturbation

  • Link enhancer accessibility to ZNF207-dependent gene regulation

• CUT&Tag/CUT&RUN at Single-Cell Level:

  • Profile ZNF207 binding sites with cellular resolution

  • Identify differential binding patterns in subpopulations

  • Map co-occupancy with OCT4/SOX2 in individual stem cells

• Spatial Transcriptomics:

  • Visualize ZNF207 expression in tissue context

  • Map spatial relationships between ZNF207-expressing cells and immune cells

  • Correlate expression with microenvironmental niches

Integrated Analysis Approaches:

Disease-Specific Applications:

• Cancer Heterogeneity:

  • Profile ZNF207 in distinct tumor cell subpopulations

  • Map ZNF207 expression in relation to cancer stem cells

  • Correlate with immunotherapy response patterns at single-cell level

• Developmental Disorders:

  • Analyze embryonic development with ZNF207 mutations/variants

  • Map consequences on cell fate decisions

  • Track effects on OCT4/SOX2 network at single-cell resolution