ZNF207 Antibody

What is ZNF207 and why is it significant in research?

ZNF207 (Zinc Finger Protein 207) is a nuclear protein that functions as a transcription factor with significant roles in multiple biological processes. Research highlights several important functions:

• Acts as a potential immunosuppressive target in hepatocellular carcinoma (HCC)

• Functions in maintaining self-renewal and pluripotency in human embryonic stem cells (hESCs)

• Operates as a kinetochore- and microtubule-binding protein (BuGZ) that plays a key role in spindle assembly during cell division

• Shows consistent upregulation across multiple cancer types, particularly in liver cancer

The protein contains two C2H2-type zinc fingers and has a calculated molecular weight of 51 kDa . Its significance lies in its emerging role in cancer progression, immune modulation, and stem cell biology, making it an increasingly important target for advanced research.

How do I select the appropriate ZNF207 antibody for my experiment?

Selection of the appropriate ZNF207 antibody requires consideration of several experimental parameters:

For Western blotting, most suppliers recommend dilutions of 1:500-1:2000 . For immunoprecipitation, 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate is typically recommended . For immunohistochemistry, dilutions of 1:25-1:100 have been validated .

Review the vendor's validation data for your specific application, particularly observing which cell lines have been successfully used (e.g., HepG2 and HeLa cells are frequently used for validating ZNF207 antibodies) .

What are the optimal conditions for Western blot detection of ZNF207?

For optimal Western blot detection of ZNF207:

Sample preparation:

• Use appropriate lysis buffers compatible with nuclear proteins

• Include protease inhibitors to prevent degradation

• Normalize protein concentration before loading

Running conditions:

• Use 10-12% SDS-PAGE gels for optimal resolution around 51 kDa

• Load 20-30 μg of total protein per lane

Antibody conditions:

• Primary antibody: Dilute ZNF207 antibody at 1:500-1:2000 in blocking buffer

• Secondary antibody: HRP-conjugated anti-rabbit IgG should be diluted 1:50,000-100,000

• Incubate primary antibody overnight at 4°C for best results

Detection:

• Expected band size: 51 kDa (observed molecular weight)

• Note: The actual band might not be consistent with the expected size due to post-translational modifications or splice variants

• Positive controls: HepG2 and HeLa cell lysates are recommended

If signal is weak, consider extended exposure times or signal enhancement systems. For specific protocols, vendors like Proteintech provide optimized product-specific Western blot protocols for their ZNF207 antibodies .

How can I optimize ChIP experiments targeting ZNF207 binding sites?

Optimizing ChIP experiments for ZNF207 requires attention to several critical parameters:

CrossLinking and Chromatin Preparation:

• Use 1% formaldehyde for 10 minutes at room temperature for effective crosslinking

• Sonicate chromatin to fragments of 200-500 bp for optimal resolution

• Validate sonication efficiency via agarose gel electrophoresis

Immunoprecipitation:

• Use 1-5 μg of ZNF207 antibody per ChIP reaction

• Include appropriate controls (IgG negative control, histone H3 positive control)

• Pre-clear chromatin with protein A+G magnetic beads to reduce background

Target Site Considerations:

• ZNF207 has been shown to bind near transcription start sites (TSS) with highest frequency

• A previously unknown DNA binding motif for ZNF207 has been identified and verified using EMSA

• ZNF207 frequently co-localizes with OCT4/SOX2 binding sites in stem cells

Validation:

• Perform qPCR analysis on known ZNF207 target regions

• According to published research, OCT4 enhancer regions have been validated as ZNF207 binding sites

• Consider preparing libraries for ChIP-seq to identify genome-wide binding patterns

In HCC research, ChIP-qPCR assays using ZNF207 antibody (Invitrogen #703747) have successfully demonstrated binding of ZNF207 to target genes, following the SimpleChIP Plus Enzymatic Chromatin IP Kit protocol .

What are the key considerations for immunohistochemical detection of ZNF207 in tissue samples?

For successful immunohistochemical detection of ZNF207 in tissue samples:

Tissue Preparation:

• Use 4-10% formalin-fixed, paraffin-embedded (FFPE) sections (4-5 μm thickness)

• Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• For frozen sections, fix in cold acetone for 10 minutes before staining

Staining Protocol:

• Block endogenous peroxidase activity with 3% H₂O₂

• Use protein blocking solution to reduce non-specific binding

• Apply ZNF207 antibody at dilutions of 1:25-1:100

• Incubate overnight at 4°C for optimal binding

• Use appropriate detection systems (HRP/DAB or fluorescent)

Controls and Interpretation:

• Include positive control tissues (e.g., human liver cancer tissue for ZNF207)

• ZNF207 shows primarily nuclear localization

• In HCC tissues, ZNF207 expression has been correlated with immune cell infiltration patterns

Validation:

• Multiple independent studies have verified ZNF207 expression in human cervical cancer samples

• Expression patterns may vary across different cancer types; increased expression has been documented in liver cancer and glioma

For quantification, consider using H-scoring or digital image analysis systems to objectively measure staining intensity and distribution in tissue samples.

Why might I observe multiple bands or unexpected molecular weights when detecting ZNF207 by Western blot?

Observing multiple bands or unexpected molecular weights for ZNF207 may occur for several reasons:

Alternative Splicing:

• ZNF207 exists in multiple isoforms due to alternative splicing events; three isoforms have been documented

• A distinct isoform of ZNF207 has been found specifically in human embryonic stem cells

• Different isoforms may perform distinct functions during cellular differentiation

Post-translational Modifications:

• Phosphorylation, ubiquitination, or other modifications can alter migration patterns

• These modifications may be cell type-specific or condition-dependent

Technical Considerations:

• Sample preparation methods (denaturing conditions, reducing agents) can affect observed molecular weight

• Incomplete denaturation can result in oligomers or protein complexes

• Proteolytic degradation may produce fragments

According to vendor literature: "The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size. If a protein in a sample has different modified forms at the same time, multiple bands may be detected on the membrane."

To troubleshoot:

• Use freshly prepared samples with protease inhibitors

• Compare different reducing and denaturing conditions

• Include positive controls (HepG2 or HeLa cell lysates)

• Consider isoform-specific antibodies if studying a particular variant

How do I reconcile contradictory findings regarding ZNF207 expression and function in different cell types?

Reconciling contradictory findings regarding ZNF207 expression and function requires systematic analysis:

Expression Variation Across Cell Types:

• ZNF207 shows tissue-specific expression patterns; higher expression has been documented in liver cancer compared to normal tissues

• Expression levels may correlate with different physiological states (e.g., stem cell maintenance vs. differentiation)

Functional Variation and Mechanisms:

• In HCC, ZNF207 functions as an immunosuppressive factor through the MAPK-chemokine axis and regulates indoleamine 2,3-dioxygenase 1 (IDO1)

• In embryonic stem cells, ZNF207 partners with OCT4/SOX2 to maintain pluripotency while simultaneously controlling commitment toward ectoderm

• As BuGZ, it functions in spindle assembly during mitosis through phase transition mechanisms

Methodological Approach to Reconciliation:

• Compare experimental conditions: Cell types, culture conditions, and assay methods may influence results

• Isoform analysis: Different isoforms may predominate in different contexts; "A distinct isoform of ZNF207 functions in hESCs at the nexus that balances pluripotency and differentiation to ectoderm"

• Multi-omics integration: Combine transcriptomic, proteomic, and functional data to build comprehensive models

• Context-dependent regulation: Consider upstream regulators like hypoxia, which has been shown to induce ZNF207 upregulation in HCC

When contradictory findings are observed, carefully document the experimental context, including cell lines, antibody clones, detection methods, and cell state. This contextual information is critical for meaningful comparison across studies.

How can ZNF207 antibodies be used to investigate its role in cancer immunotherapy resistance?

ZNF207 antibodies can be leveraged to investigate immunotherapy resistance through several methodological approaches:

Translational Research Applications:

• Patient stratification: Multiple studies indicate that ZNF207 expression correlates with response to anti-PD1 therapy; "Patients with lower ZNF207 expression were more sensitive to antiprogrammed cell death protein 1 (PD1) therapy"

• Biomarker development: ZNF207 expression analysis in pre-treatment biopsies using IHC or Western blot

Mechanistic Investigations:

• Immune microenvironment analysis:

  • Multiplex immunofluorescence with ZNF207 antibody co-staining with immune cell markers

  • ZNF207 positively correlates with immunosuppressive cell markers (FOXP3, CD11b, CD68) and negatively with cytotoxic T cells (CD8A)

• Molecular pathway analysis:

  • ZNF207 transcriptionally regulates indoleamine 2,3-dioxygenase 1 (IDO1)

  • ChIP-qPCR using ZNF207 antibodies can quantify binding to the IDO1 promoter

  • Co-immunoprecipitation to identify interacting partners in immunotherapy-resistant models

• Functional assays:

  • Cytokine array analysis comparing ZNF207-high vs. ZNF207-silenced cancer models

  • T-cell co-culture experiments to measure cytotoxicity associated with ZNF207 expression levels

Combinatorial Therapy Development:
Research indicates that "silencing ZNF207 could be beneficial to anti-PD1 combination therapy" . To investigate this:

• Use ZNF207 antibodies to screen responders vs. non-responders in preclinical models

• Employ tissue microarrays with ZNF207 IHC to correlate expression with clinical outcomes

• Develop companion diagnostic approaches based on ZNF207 detection

The comprehensive pan-cancer analysis indicates ZNF207 is "a promising prognostic biomarker and immunotherapeutic target" across multiple cancer types .

What techniques can be used to study ZNF207 isoform switching during cellular differentiation?

Studying ZNF207 isoform switching during cellular differentiation requires specialized techniques:

Isoform Detection and Quantification:

• Isoform-specific RT-qPCR:

  • Design primers spanning unique exon junctions

  • Utilize TaqMan probes targeting isoform-specific sequences

  • Normalize to appropriate reference genes

• Western blotting with isoform resolution:

  • Use high-resolution SDS-PAGE (8-10%) with extended run times

  • Consider Phos-tag gels to resolve phosphorylated isoforms

  • Use antibodies targeting common and isoform-specific epitopes

• RNA-sequencing analysis:

  • Perform deep sequencing with adequate coverage for splice junction detection

  • Apply computational tools (e.g., RSEM, Kallisto) for isoform-level quantification

  • Validate with targeted RT-PCR of specific splice junctions

Functional Analysis of Isoform Switching:
Research has shown that "the distinct roles of ZNF207 during differentiation occur via isoform switching" . To study this:

• ChIP-Seq with isoform-specific antibodies:

  • Compare genomic binding sites of different isoforms

  • Analyze motif enrichment at isoform-specific binding sites

  • Correlate with gene expression changes during differentiation

• Isoform-specific knockdown and overexpression:

  • Design shRNAs or CRISPR guides targeting unique exons

  • Express individual isoforms in knockout backgrounds

  • Assess phenotypic impacts on differentiation trajectories

• Protein interaction profiling:

  • Perform co-immunoprecipitation followed by mass spectrometry

  • Compare interactomes of different ZNF207 isoforms

  • Map interaction differences to functional consequences

For stem cell differentiation studies, published research indicates a "distinct isoform of ZNF207 functions in hESCs at the nexus that balances pluripotency and differentiation to ectoderm" , suggesting careful monitoring during differentiation timecourses.

How can I investigate the dual role of ZNF207 in both immune regulation and mitotic progression?

Investigating the dual role of ZNF207 requires parallel approaches targeting its distinct functions:

Experimental Design Strategy:

Immune Regulation Assessment:

• Molecular mechanism investigation:

  • ChIP-seq to map ZNF207 binding sites on immunomodulatory genes

  • Analysis of the MAPK-chemokine axis implicated in ZNF207-mediated CD8+ T-cell chemotaxis

  • Measure kynurenine levels, which are elevated by ZNF207 regulation of IDO1

• Functional immune assays:

  • Cytokine array analysis comparing ZNF207-silenced vs. control cells

  • T-cell migration and infiltration assays

  • Analysis of T-cell exhaustion markers in co-culture systems

Mitotic Function Assessment:

• BuGZ/microtubule interaction studies:

  • Immunofluorescence microscopy to visualize ZNF207/BuGZ localization during mitosis

  • In vitro microtubule bundling assays

  • Phase transition/coacervation analysis, as "ZNF207/BuGZ undergoes phase transition or coacervation to form temperature-dependent liquid droplets"

• Kinetochore function analysis:

  • Co-immunoprecipitation with BUB3 protein

  • Assessment of chromosome alignment defects

  • Analysis of mitotic checkpoint signaling

Integrated Analysis:
To understand how these distinct functions might be regulated:

• Use phospho-specific antibodies to determine if phosphorylation states segregate functions

• Perform domain mapping through truncation mutants to identify regions required for each function

• Conduct temporal analysis across cell cycle phases and immune activation states

Research indicates that ZNF207's multiple functions may be context-dependent; as noted, it "plays a key role in spindle assembly" while also being "implicated in suppressing the HCC microenvironment" .

What are the best approaches to study ZNF207's role in the cancer–immunity cycle?

Studying ZNF207's role in the cancer-immunity cycle requires a systematic approach addressing multiple steps in this process:

Comprehensive Analysis Framework:

• Cancer-Immunity Cycle Scoring:

  • Apply computational methods to quantify each step of the cancer-immunity cycle

  • Correlate ZNF207 expression with cycle scores across patient cohorts

  • Research indicates ZNF207 negatively correlates with steps 5-7 of the cancer-immunity cycle

• Multi-modal Tissue Analysis:

  • Multiplex immunohistochemistry to co-stain ZNF207 with immune cell markers

  • Spatial transcriptomics to map ZNF207 expression relative to immune compartments

  • Digital pathology with quantitative image analysis for correlation studies

• Mechanistic Dissection:

  • ChIP-seq and RNA-seq integration to identify direct transcriptional targets

  • Cytokine profiling using proteome profiler arrays as described in the literature

  • Metabolomic analysis focusing on immunomodulatory metabolites like kynurenine

Step-Specific Experimental Approaches:

Translational Validation:

• Analyze patient cohorts receiving immunotherapy

• Stratify based on ZNF207 expression levels

• Research shows "patients with lower ZNF207 expression were more sensitive to antiprogrammed cell death protein 1 (PD1) therapy"

This systematic approach enables comprehensive characterization of ZNF207's role across all steps of the cancer-immunity cycle, providing insights for potential therapeutic interventions.

What are the critical quality control steps for validating ZNF207 antibody specificity?

Validating ZNF207 antibody specificity requires multiple complementary approaches:

Positive and Negative Controls:

• Positive cellular controls: Use cell lines with documented ZNF207 expression (HepG2, HeLa)

• Genetic controls: Compare wildtype vs. ZNF207 knockdown/knockout samples

• Tissue controls: Include tissues with known expression patterns based on RNA-seq data

Validation Methodologies:

• Western blot validation:

  • Confirm expected molecular weight (51 kDa)

  • Verify band disappearance in knockdown/knockout samples

  • Test multiple antibodies targeting different epitopes

• Immunoprecipitation-mass spectrometry:

  • Perform IP followed by MS identification of pulled-down proteins

  • Confirm ZNF207 peptides are recovered

  • Identify expected interaction partners (e.g., OCT4/SOX2 in stem cells)

• Immunofluorescence co-localization:

  • Verify nuclear localization pattern

  • Co-stain with markers of known ZNF207 compartments

  • Compare staining patterns across multiple antibody clones

• Cross-reactivity assessment:

  • Test antibody performance across species (human, mouse, rat)

  • Check for signal in tissues known to lack ZNF207 expression

  • Perform peptide competition assays

Reporting Standards:

• Document complete validation information including antibody catalog numbers, lots, and dilutions

• Include all validation controls in publications

• Report any observed discrepancies between expected and observed results

Commercial antibodies typically undergo antigen affinity purification , but independent validation remains essential for research applications.

How should ZNF207 antibody storage and handling be managed to maintain optimal performance?

Proper storage and handling of ZNF207 antibodies is critical for maintaining optimal performance:

Storage Conditions:

• Most ZNF207 antibodies should be stored at -20°C for long-term stability

• For short periods (days), storage at 4°C is acceptable

• Many commercial preparations contain 50% glycerol to prevent freeze-thaw damage

• Some preparations include stabilizers like 0.02-0.09% sodium azide

Handling Guidelines:

• Aliquoting strategy:

  • Upon receipt, prepare small working aliquots to avoid repeated freeze-thaw cycles

  • Typical aliquot volumes of 10-20 μl are practical for most applications

  • Document the date of aliquoting and number of freeze-thaw cycles

• Thawing protocol:

  • Thaw antibodies completely at 4°C or on ice, never at room temperature

  • Mix gently by flicking or mild vortexing; avoid excessive agitation

  • Spin briefly after thawing to collect contents at the bottom of the tube

• Working dilution preparation:

  • Prepare fresh working dilutions on the day of experiment

  • Use high-quality, clean tubes for dilutions

  • Include stabilizing proteins (e.g., BSA) in dilution buffers

Tracking and Documentation:

• Maintain a laboratory antibody inventory system

• Record lot numbers, receipt dates, and aliquoting information

• Document performance across experiments to identify potential degradation

Stability Considerations:

• Typical shelf life is 12 months from date of receipt when properly stored

• Monitor changes in antibody performance over time

• As noted by suppliers: "Avoid multiple freeze-thaw cycles"