ZNF638 Antibody

What is ZNF638 and why is it significant for research?

ZNF638 (Zinc finger protein 638), also known as NP220 and ZFML, is a 221 kDa nuclear protein that functions as a transcriptional coregulator in several biological processes. It has gained research significance due to its roles in:

• Adipocyte differentiation via PPARγ induction in cooperation with CCAAT/enhancer binding proteins (C/EBPs)

• Epigenetic silencing of endogenous retroelements through recruitment of the HUSH complex

• Regulation of triglyceride metabolism via ANGPTL8 in an estrogen-dependent manner

• Modulation of antiviral immune responses in cancer, particularly glioblastoma

ZNF638 is localized in nuclear bodies enriched with splicing factors, suggesting potential involvement in RNA processing mechanisms . Its multifunctional nature makes it a valuable target for researchers studying transcriptional regulation, immunomodulation, and metabolic processes.

What are the key differences between polyclonal and recombinant ZNF638 antibodies?

The choice between polyclonal and recombinant ZNF638 antibodies depends on your specific experimental requirements:

For initial characterization studies, polyclonal antibodies may provide broader epitope recognition. For precise localization or quantification experiments requiring consistent results across multiple studies, recombinant antibodies offer superior reproducibility and specificity .

What molecular weight should I expect when detecting ZNF638 by Western blot?

While the calculated molecular weight of ZNF638 is 221 kDa based on amino acid sequence, the observed molecular weight in Western blot applications typically ranges from 270-300 kDa . This discrepancy is important to note when interpreting your results.

The higher apparent molecular weight is likely due to:

• Post-translational modifications (particularly phosphorylation)

• The presence of arginine-serine (RS) domains that affect protein migration

• Protein-protein interactions that persist despite denaturing conditions

When performing Western blot experiments, use appropriate molecular weight markers that extend to 300 kDa. Additionally, include positive control lysates from cells known to express ZNF638 (HEK-293T, HeLa, HepG2, or Jurkat cells) to validate band identity .

How should I optimize Western blot protocols for detecting ZNF638?

Detecting ZNF638 via Western blot requires careful optimization due to its high molecular weight and variable expression levels across cell types:

Recommended Western Blot Protocol:

• Sample Preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • Load 20-30 μg of total protein per lane

  • Heat samples at 95°C for 5-10 minutes in reducing conditions

• Gel Electrophoresis:

  • Use 4-12% gradient gels to accommodate the high molecular weight

  • Run at 100V until the dye front reaches the bottom

• Transfer:

  • Employ wet transfer method (100V for 90 minutes or 30V overnight at 4°C)

  • Use PVDF membrane (0.45 μm pore size) rather than nitrocellulose

• Antibody Incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Primary antibody dilutions:

    • Polyclonal antibodies (e.g., 31300-1-AP): 1:1000-1:8000

    • Recombinant antibodies (e.g., 84359-3-RR): 1:5000-1:50000

  • Incubate primary antibody overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5000 for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence with extended exposure times (1-5 minutes)

Positive Controls: HEK-293T, HeLa, HepG2, and Jurkat cells all express detectable levels of ZNF638 .

What are the best approaches for immunofluorescence detection of ZNF638?

ZNF638 predominantly localizes to nuclear bodies associated with splicing factors, making it an interesting target for immunofluorescence studies:

Optimized Immunofluorescence Protocol:

• Cell Preparation:

  • Culture cells on glass coverslips to 70-80% confluency

  • Fix with 4% formaldehyde solution for 15-20 minutes at room temperature

• Permeabilization and Blocking:

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

  • Block with 1-5% BSA in PBS for 30-60 minutes

• Antibody Incubation:

  • Primary antibody dilution: 1:200-1:800 for recombinant antibodies (84359-3-RR)

  • Incubate overnight at 4°C in a humidified chamber

  • Secondary antibody: Alexa Fluor 488-labeled anti-rabbit at 1:500 for 1 hour at room temperature

• Nuclear Counterstaining:

  • DAPI (1 μg/ml) for 5 minutes

  • Mount with anti-fade mounting medium

For co-localization studies, consider dual staining with markers of nuclear speckles or splicing factors, as ZNF638 has been shown to co-localize with these structures .

How can I validate ZNF638 antibody specificity for my experimental system?

Validating antibody specificity is critical for ensuring reliable experimental results, especially for less characterized proteins like ZNF638:

Comprehensive Validation Strategy:

• Genetic Validation:

  • Perform siRNA or shRNA knockdown of ZNF638 in your target cells

  • Compare antibody reactivity between control and knockdown samples by Western blot and immunofluorescence

  • A specific antibody will show significantly reduced signal in knockdown cells

• Overexpression Validation:

  • Transfect cells with a tagged ZNF638 construct (FLAG-ZNF638 or GFP-ZNF638)

  • Perform parallel detection with anti-tag antibody and ZNF638 antibody

  • Signal co-localization confirms antibody specificity

• Peptide Competition:

  • Pre-incubate the antibody with excess immunizing peptide

  • The specific signal should be blocked or substantially reduced

• Multi-antibody Concordance:

  • Compare results using multiple antibodies targeting different epitopes of ZNF638

  • Consistent detection patterns across antibodies increase confidence in specificity

• Molecular Weight Verification:

  • Confirm detection at the expected molecular weight (270-300 kDa)

  • Be cautious of additional bands that may represent isoforms or degradation products

For advanced validation, consider using CRISPR/Cas9-mediated knockout of ZNF638 as the gold standard negative control .

How can I use ZNF638 antibodies to study its role in regulating antiviral immune responses?

Recent research has revealed ZNF638 as a regulator of endogenous retroelement silencing and antiviral immune responses, particularly in cancer contexts:

Experimental Approach:

• Analyzing ZNF638-HUSH Complex Interactions:

  • Perform co-immunoprecipitation with ZNF638 antibody followed by Western blot detection of HUSH complex components (TASOR, MPP8, SETDB1)

  • Expected outcome: ZNF638 pulldown should co-precipitate HUSH complex proteins

• Assessing H3K9 Trimethylation Levels:

  • Use ChIP-seq with H3K9me3 antibodies in control vs. ZNF638 knockdown cells

  • Analyze enrichment at endogenous retroelement loci

  • Expected outcome: ZNF638 knockdown should reduce H3K9me3 marks at these regions

• Measuring dsRNA Expression:

  • Perform dsRNA immunoprecipitation using J2 antibody in control vs. ZNF638 knockdown cells

  • Quantify by qPCR specific retroelements (LINE-1, Alus, LTRs)

  • Expected outcome: ZNF638 knockdown should increase dsRNA levels

• Downstream Signaling Analysis:

  • Assess activation of dsRNA sensing pathways (RIG-I, MDA5, TLR3) and IRF3 phosphorylation by Western blot

  • Measure type I interferon production by ELISA

  • Expected outcome: ZNF638 knockdown should enhance these antiviral signaling pathways

• Immune Checkpoint Expression:

  • Analyze PD-L1 levels by flow cytometry or immunofluorescence in ZNF638-depleted cells

  • Expected outcome: ZNF638 knockdown should increase PD-L1 expression

This experimental framework allows for comprehensive analysis of how ZNF638 regulates the antiviral immune response cascade in your cell type of interest.

What approaches can be used to study ZNF638's role in adipocyte differentiation?

ZNF638 functions as a transcriptional coregulator of adipogenesis through interactions with C/EBPs and regulation of PPARγ expression:

Methodological Approach:

• Protein-Protein Interaction Analysis:

  • Perform co-immunoprecipitation of ZNF638 with C/EBP factors

  • Use 5 μg of FLAG-ZNF638 and 1 μg of C/EBPβ in transfected HEK-293 cells

  • Immunoprecipitate with anti-FLAG M2 affinity gel and detect interaction by Western blot

• Domain Mapping Studies:

  • Generate GFP-tagged ZNF638 fragments (amino acids 1-610, 607-1118, 1110-1780, 1773-1927)

  • Assess which domains interact with C/EBP factors using GFP-Trap beads

  • The domain spanning amino acids 607-1118 is particularly relevant for these interactions

• Transcriptional Regulation Analysis:

  • Use luciferase reporter assays with C/EBP-luciferase or PPARγ2-luciferase constructs

  • Co-transfect with ZNF638 expression plasmids (100-150 ng) and C/EBP factors (10 ng)

  • Measure luciferase activity 48 hours post-transfection

• Functional Differentiation Assays:

  • Transfect 10T1/2 preadipocytes with ZNF638 expression constructs or knockdown reagents

  • Induce differentiation with standard MDI medium plus 100 nm rosiglitazone

  • Assess adipogenesis through Oil Red O staining and expression of adipocyte markers

These approaches provide a comprehensive framework for dissecting ZNF638's molecular mechanisms in adipocyte differentiation.

How can I design experiments to investigate ZNF638's potential as an immunotherapy target in cancer?

Based on recent findings showing ZNF638 as a regulator of antiviral immune responses and potential immunotherapy target in glioblastoma, researchers can design experiments to explore this further:

Experimental Framework:

• Relationship Between ZNF638 Expression and Immune Landscape:

  • Analyze correlation between ZNF638 and immune cell infiltration markers using public datasets (TCGA, TIMER)

  • Expected outcome: ZNF638 expression should negatively correlate with CD8+ T-cell infiltration

• In Vitro Immune Response Modulation:

  • Generate stable ZNF638 knockdown in cancer cell lines

  • Co-culture with immune cells (T cells, dendritic cells)

  • Measure:

    • PD-L1 expression in cancer cells (flow cytometry)

    • T cell activation markers (CD69, CD25)

    • Cytokine production (IFN-γ, IL-2)

• Syngeneic Mouse Models:

  • Establish orthotopic cancer models with ZNF638-knockdown cells

  • Treat with immune checkpoint inhibitors (anti-PD-L1)

  • Monitor:

    • Tumor growth

    • Survival outcomes

    • Immune infiltration by flow cytometry and immunohistochemistry

    • Cytokine profiles in tumor microenvironment

• Analysis of Clinical Samples:

  • Assess ZNF638 expression in patient samples responding to immunotherapy versus non-responders

  • Correlate with:

    • dsRNA expression

    • PD-L1 levels

    • CD8+ T-cell infiltration

    • Treatment outcomes

This approach enables comprehensive evaluation of ZNF638 as an immunotherapy target across preclinical models and clinical contexts.

What are common issues when working with ZNF638 antibodies and how can they be resolved?

Working with high molecular weight proteins like ZNF638 presents several technical challenges:

For particularly challenging applications, consider using a combination of antibodies targeting different epitopes to confirm results.

How should I interpret discrepancies in ZNF638 detection between different experimental techniques?

Researchers often encounter variations in ZNF638 detection across different methods. Understanding these discrepancies is crucial for accurate data interpretation:

Common Discrepancies and Interpretations:

• Western Blot vs. Immunofluorescence:

  • Observation: Strong Western blot signal but weak immunofluorescence staining

  • Interpretation: Epitope masking in native conformation or localization in specific nuclear compartments

  • Verification: Try different fixation methods (paraformaldehyde vs. methanol) or antibodies targeting different epitopes

• Predicted vs. Observed Molecular Weight:

  • Observation: Calculated MW is 221 kDa but observed is 270-300 kDa

  • Interpretation: Post-translational modifications affect migration

  • Verification: Treat lysates with phosphatase to confirm if phosphorylation contributes to size increase

• RNA vs. Protein Expression Levels:

  • Observation: High mRNA levels with low protein detection

  • Interpretation: Post-transcriptional regulation or protein instability

  • Verification: Treat cells with proteasome inhibitors to assess protein stability

• Cell-Type Specific Variations:

  • Observation: Variable detection across cell lines

  • Interpretation: Tissue-specific expression or regulation

  • Verification: Compare with transcriptomic data from databases like ARCHS4

• Nuclear vs. Cytoplasmic Fractions:

  • Observation: Different subcellular localization patterns

  • Interpretation: Stimulus-dependent translocation or isoform-specific localization

  • Verification: Perform subcellular fractionation alongside immunofluorescence

When encountering discrepancies, it's advisable to employ complementary methods and antibodies to build a more complete understanding of ZNF638 biology in your experimental system.

How is ZNF638 research advancing our understanding of cancer immunotherapy?

Recent research has uncovered a previously unknown role for ZNF638 in regulating antiviral immune responses and cancer immunotherapy sensitivity:

Key Research Findings:

• Epigenetic Silencing Mechanism:

  • ZNF638 recruits the HUSH complex to endogenous retroelements

  • This leads to repressive H3K9me3 marks and silencing of retroelements

  • Knockdown of ZNF638 decreases H3K9 trimethylation and increases cytosolic dsRNA

• Antiviral Immune Pathway Activation:

  • ZNF638 knockdown activates dsRNA sensing pathways (RIG-I, MDA5, TLR3)

  • This triggers downstream signaling via MAVS, TRAF3, TBK1, and IRF3 phosphorylation

  • The result is increased expression of type I interferons and proinflammatory cytokines

• Immunotherapy Sensitization:

  • ZNF638 depletion significantly increases PD-L1 expression on cancer cells

  • In syngeneic mouse models, ZNF638 knockdown enhances response to PD-L1 checkpoint inhibitors

  • This leads to dramatically improved survival and reduced tumor volumes (90-fold smaller)

• Clinical Correlation:

  • Low ZNF638 expression correlates with better clinical response to immunotherapy

  • This has been observed in both recurrent glioblastoma and melanoma patients

  • ZNF638 expression patterns could potentially serve as a biomarker for immunotherapy response

These findings suggest that targeting ZNF638 could be a novel strategy to enhance immunotherapy efficacy, particularly in immunologically "cold" tumors like glioblastoma.

What are the latest methodological advances in studying ZNF638's molecular interactions?

Recent technical innovations have expanded our ability to study ZNF638's complex interactions and functions:

Advanced Methodological Approaches:

• Proximity-Based Interaction Mapping:

  • BioID or TurboID fusion proteins allow identification of proximal interactors

  • APEX2-based proximity labeling can capture transient interactions

  • These approaches have identified previously unknown ZNF638 binding partners beyond the HUSH complex

• ChIP-seq and CUT&RUN Applications:

  • These techniques map genome-wide binding profiles of ZNF638

  • They reveal preferential binding to retroelement loci and regulatory regions

  • Integration with H3K9me3 profiles establishes direct links to epigenetic silencing

• Single-Cell Transcriptomics:

  • Analysis of ZNF638 expression across cell types in heterogeneous tissues

  • Correlation with immune infiltration signatures at single-cell resolution

  • Identification of cell-specific roles in different microenvironmental contexts

• CRISPR Screening Approaches:

  • Genome-wide CRISPR screens identifying genes that synergize with ZNF638 inhibition

  • Domain-focused CRISPR screens to map functional regions of the protein

  • These screens have uncovered synthetic lethal interactions relevant to cancer therapy

• Patient-Derived Organoid Models:

  • 3D culture systems that better recapitulate in vivo biology

  • Allow testing of ZNF638 manipulation in more physiologically relevant contexts

  • Enable evaluation of immune cell interactions in co-culture systems

These methodological advances are accelerating our understanding of ZNF638's multifaceted roles across different biological contexts and disease states.

What is the potential of ZNF638 as a therapeutic target beyond cancer applications?

While recent attention has focused on ZNF638's role in cancer immunotherapy, emerging research suggests broader therapeutic applications:

Expanding Therapeutic Horizons:

• Metabolic Disorders:

  • ZNF638 regulates adipocyte differentiation through PPARγ pathways

  • It also modulates triglyceride metabolism via ANGPTL8 in an estrogen-dependent manner

  • These functions suggest potential applications in obesity and metabolic syndrome

• Autoimmune Conditions:

  • ZNF638's regulation of endogenous retroelements and antiviral responses may impact autoimmunity

  • Retroelement expression has been linked to several autoimmune disorders

  • Modulating ZNF638 could potentially rebalance immune responses in these conditions

• Developmental Programming:

  • ZNF638's interactions with key transcriptional regulators like C/EBPs suggest roles in cellular differentiation

  • This could be relevant for regenerative medicine applications

  • It may offer novel approaches to direct stem cell differentiation into specific lineages

• Viral Infections:

  • The role of ZNF638 in dsRNA sensing and antiviral responses extends to infectious diseases

  • Targeting ZNF638 might enhance innate immune responses to viral challenges

  • This could complement existing antiviral therapies with immune-stimulating effects

• Neurological Disorders:

  • ZNF638's expression in neural tissues and role in epigenetic regulation

  • Potential implications for neurodevelopmental and neurodegenerative conditions

  • May offer novel approaches to address epigenetic dysregulation in these disorders