ZNF544 Antibody

What is ZNF544 and what cellular functions does it serve?

ZNF544 (Zinc finger protein 544) is a member of the Kruppel C2H2-type zinc-finger protein family that functions primarily as a transcription factor. It is a 715 amino acid protein containing 13 C2H2-type zinc fingers and 1 KRAB domain . ZNF544 localizes to the nucleus and plays crucial roles in controlling gene expression and cellular function through DNA-binding transcription activator activity and RNA polymerase II-specific regulation . As a transcription factor, it is implicated in various cellular processes including gene regulation and cell differentiation, making it particularly relevant to developmental biology and cancer research . The protein contains specific DNA-binding domains that enable it to recognize and bind to target DNA sequences, thereby modulating the expression of specific genes within cellular networks.

What types of ZNF544 antibodies are currently available for research applications?

The current research landscape offers several well-characterized ZNF544 antibodies suitable for different experimental applications:

These antibodies are typically affinity-purified from rabbit antiserum using epitope-specific immunogens, with purity levels exceeding 95% as determined by SDS-PAGE . Most are supplied as unconjugated antibodies in buffered aqueous glycerol solutions and should be stored at -20°C to maintain reactivity and specificity .

How should I optimize Western blot conditions for ZNF544 detection?

For optimal Western blot detection of ZNF544, consider the following methodological approach:

• Sample preparation: Prepare protein lysates from appropriate human or mouse tissues/cells under conditions that preserve protein integrity. Nuclear extraction protocols are particularly important as ZNF544 predominantly localizes to the nucleus .

• Protein loading: Load 20-40 μg of total protein per lane, ensuring equal loading across samples using housekeeping protein controls (e.g., β-actin, GAPDH).

• Gel separation: Use 8-10% SDS-PAGE gels for optimal separation as ZNF544 has a calculated molecular weight of approximately 82 kDa .

• Transfer parameters: Transfer proteins to nitrocellulose or PVDF membranes using standard protocols (e.g., 100V for 1 hour or 30V overnight).

• Blocking: Block non-specific binding sites with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute ZNF544 antibody at 1:500 to 1:2000 in blocking buffer (as recommended for CAB7366) . Incubate overnight at 4°C with gentle agitation.

• Detection method: Use appropriate HRP-conjugated secondary antibodies and enhanced chemiluminescence for visualization.

• Expected band: Look for a specific band at approximately 82 kDa .

• Validation controls: Include positive control samples such as A-431 cells or mouse kidney tissue, which have been confirmed to express ZNF544 .

What are the key considerations for immunohistochemistry (IHC) experiments with ZNF544 antibodies?

When performing IHC with ZNF544 antibodies, researchers should consider these methodological details:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) sections or frozen sections depending on the specific antibody recommendations.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) to expose antigenic sites that may be masked during fixation.

• Antibody dilution: For optimal staining, use a dilution range of 1:200-1:500 for IHC applications as recommended for the HPA002732 antibody .

• Incubation conditions: Incubate primary antibody overnight at 4°C or for 1-2 hours at room temperature in a humidified chamber.

• Detection system: Use a polymer-based detection system for enhanced sensitivity and reduced background.

• Counterstaining: Apply hematoxylin for nuclear counterstaining, which helps localize ZNF544 staining relative to cellular structures.

• Positive controls: Include tissues known to express ZNF544, such as specific normal human tissues that have been validated through the Human Protein Atlas project .

• Negative controls: Include sections where primary antibody is omitted or replaced with non-immune serum from the same species.

• Expected pattern: Look for nuclear localization of ZNF544 staining, consistent with its function as a transcription factor .

How can I investigate ZNF544's role in transcriptional regulation networks?

To investigate ZNF544's role in transcriptional regulation, implement the following comprehensive approach:

• Chromatin Immunoprecipitation (ChIP):

  • Use ZNF544 antibodies to perform ChIP experiments followed by sequencing (ChIP-seq) to identify genome-wide binding sites.

  • Start with crosslinking cells using 1% formaldehyde, followed by sonication to shear chromatin into 200-500 bp fragments.

  • Immunoprecipitate using 2-5 μg of ZNF544 antibody per reaction.

  • Analyze enriched regions to identify consensus binding motifs and associated genes.

• Gene expression analysis after ZNF544 modulation:

  • Implement CRISPR-Cas9 knockout or siRNA knockdown of ZNF544.

  • Perform RNA-seq to identify differentially expressed genes.

  • Compare ChIP-seq and RNA-seq data to identify direct transcriptional targets.

• Protein-protein interaction studies:

  • Use co-immunoprecipitation with ZNF544 antibodies followed by mass spectrometry to identify interaction partners.

  • Confirm interactions with orthogonal methods such as proximity ligation assay (PLA).

  • Map interaction domains through mutagenesis studies.

• KRAB domain functional analysis:

  • Create domain-specific deletions to assess the role of the KRAB domain in recruiting co-repressors like KAP1.

  • Analyze changes in heterochromatin formation using ChIP for histone modifications (H3K9me3).

This multi-dimensional approach will provide insights into how ZNF544 contributes to transcriptional networks and gene regulation within specific cellular contexts.

What is the current understanding of ZNF544's role in cancer biology and potential therapeutic implications?

Recent research has revealed important connections between ZNF544 and cancer biology:

• Expression patterns in cancer: ZNF544 belongs to a cluster of primate-specific KRAB zinc finger proteins that have been found upregulated in certain cancers, including diffuse large B-cell lymphoma (DLBCL), with associations to poor prognosis and increased copy-number alterations .

• Functional significance: Evidence suggests that ZNF544 and related KZFPs may protect cancer cells by:

  • Maintaining heterochromatin at transposable elements (TEs)

  • Preventing excessive replicative stress associated with genome-wide alterations

  • Safeguarding cancer cells against cell-intrinsic inflammatory responses

  • Potentially contributing to immune evasion mechanisms

• Experimental approaches to study ZNF544 in cancer:

  • Analyze expression levels across cancer types using transcriptomic databases

  • Perform survival analyses correlating ZNF544 expression with patient outcomes

  • Investigate phenotypic changes following ZNF544 depletion in cancer cell models

  • Examine effects on heterochromatin distribution, replicative stress, and inflammatory response pathways

• Methodological considerations:

  • Use cell proliferation assays (such as MTT) after ZNF544 depletion to assess functional impact

  • Implement immunofluorescence staining to detect heterochromatin changes

  • Monitor markers of replicative stress and cGAS-STING pathway activation

  • Assess immune-related phenotypes such as HLA-I expression and susceptibility to immune detection

This evidence suggests that targeting ZNF544 could potentially sensitize cancer cells to immune surveillance mechanisms, representing a novel avenue for therapeutic development.

How do I address non-specific binding issues when using ZNF544 antibodies?

When encountering non-specific binding with ZNF544 antibodies, implement this systematic troubleshooting approach:

• Antibody validation and selection:

  • Verify antibody specificity through published validation data, particularly from resources like the Human Protein Atlas .

  • Consider antibodies that have been affinity-purified (>95% purity by SDS-PAGE) .

  • Review the immunogen sequence to ensure it targets unique regions of ZNF544.

• Optimization of blocking conditions:

  • Test different blocking agents (BSA, non-fat dry milk, normal serum) at varying concentrations (3-5%).

  • Extend blocking time to 2 hours at room temperature.

  • Add 0.1-0.3% Triton X-100 or 0.05% Tween-20 to reduce hydrophobic interactions.

• Antibody dilution optimization:

  • Perform titration experiments with serial dilutions (e.g., 1:200, 1:500, 1:1000, 1:2000).

  • For immunofluorescence, start with the recommended range of 0.25-2 μg/mL .

  • For Western blot, begin with 1:500-1:2000 dilutions .

• Incubation conditions:

  • Try reducing primary antibody incubation temperature (4°C instead of room temperature).

  • Extend washing steps (5 x 5 minutes with gentle agitation).

  • Consider adding low concentrations of competing proteins to reduce non-specific interactions.

• Cross-reactivity assessment:

  • Perform peptide competition assays using the immunizing peptide.

  • Include knockout/knockdown controls to confirm signal specificity.

  • Test antibody on samples from different species to assess cross-reactivity claims.

Implementation of these methodological refinements should significantly improve signal-to-noise ratio and enhance the specificity of ZNF544 detection across experimental platforms.

What are the best approaches for validating ZNF544 antibody specificity?

To rigorously validate ZNF544 antibody specificity, researchers should implement multiple complementary approaches:

• Genetic knockout/knockdown validation:

  • Generate CRISPR-Cas9 knockout cell lines for ZNF544.

  • Alternatively, use siRNA or shRNA-mediated knockdown.

  • Compare antibody signal between wild-type and knockout/knockdown samples across multiple applications (WB, IHC, IF).

  • A genuine ZNF544 antibody will show significantly reduced or absent signal in knockout/knockdown samples.

• Overexpression studies:

  • Transfect cells with ZNF544 expression constructs (with and without tags).

  • Confirm increased signal intensity in transfected versus non-transfected cells.

  • Use tagged constructs to co-stain with anti-tag antibodies and analyze signal overlap.

• Epitope mapping and peptide competition:

  • Identify the specific epitope sequence recognized by the antibody.

  • Perform pre-absorption experiments by incubating the antibody with excess immunizing peptide.

  • Observe signal reduction in pre-absorbed samples compared to non-absorbed controls.

• Orthogonal detection methods:

  • Compare results from different antibodies targeting distinct epitopes of ZNF544.

  • Correlate protein detection with mRNA expression data in the same samples.

  • Use mass spectrometry to confirm the identity of immunoprecipitated proteins.

• Cross-platform validation:

  • Test antibody performance across multiple techniques (WB, IP, IHC, IF).

  • Look for consistent molecular weight detection (82 kDa) in Western blots .

  • Confirm expected subcellular localization (nuclear) in immunofluorescence and IHC .

These comprehensive validation approaches ensure that experimental observations attributed to ZNF544 are specific and reproducible, establishing a solid foundation for subsequent research.

How can ZNF544 antibodies be used to study its role in heterochromatin regulation and genomic stability?

Recent research has identified links between ZNF544 and heterochromatin maintenance . To investigate this connection:

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Use ZNF544 antibodies to immunoprecipitate associated chromatin.

  • Analyze binding patterns in relation to heterochromatin markers (H3K9me3, H3K27me3).

  • Focus particularly on transposable element regions, where KZFPs are known to bind.

  • Compare binding patterns in normal versus cancer cells to identify disease-specific alterations.

• Co-immunoprecipitation with chromatin modifiers:

  • Implement co-IP protocols using ZNF544 antibodies to pull down associated proteins.

  • Analyze interactions with known heterochromatin regulators (KAP1, SETDB1, HP1).

  • Validate interactions through reciprocal immunoprecipitation and proximity ligation assays.

• Replicative stress assessment:

  • Combine ZNF544 antibody staining with markers of replicative stress (γH2AX, pRPA).

  • Implement immunofluorescence co-localization analysis following ZNF544 depletion.

  • Monitor changes in R-loop formation and DNA damage accumulation.

• Genomic instability measurements:

  • Use ZNF544 antibodies in combination with FISH techniques to assess structural changes.

  • Implement metaphase spread analysis after ZNF544 modulation.

  • Correlate ZNF544 expression/binding with copy number variations in cancer genomes.

• Methodological approach for heterochromatin analysis:

  • Perform microscopy-based assays combining ZNF544 staining with heterochromatin markers.

  • Implement high-content imaging to quantify heterochromatin distribution.

  • Use DNA methylation analysis to correlate ZNF544 binding with epigenetic modifications.

These approaches provide a comprehensive framework for understanding how ZNF544 contributes to genome integrity through heterochromatin maintenance, with particular relevance to cancer biology and cell fate decisions.

What are the methodological considerations for studying ZNF544's potential role in immune evasion mechanisms in cancer?

Based on emerging research indicating that KZFPs including ZNF544 may contribute to immune evasion in cancer , researchers should consider these methodological approaches:

• Immune phenotyping after ZNF544 modulation:

  • Deplete ZNF544 in cancer cell lines using RNAi or CRISPR techniques.

  • Analyze changes in surface expression of immune-related molecules (HLA-I, PD-L1, etc.) by flow cytometry.

  • Compare results across multiple cancer cell types to identify common vs. context-specific effects.

• Inflammatory response monitoring:

  • Implement qRT-PCR panels targeting interferon-stimulated genes (ISGs) after ZNF544 knockdown.

  • Analyze secreted inflammatory cytokines using multiplex assays or ELISAs.

  • Monitor cGAS-STING pathway activation through phospho-STING and phospho-TBK1 detection.

• Co-culture assay design:

  • Establish co-culture systems with immune cells (T cells, NK cells, macrophages).

  • Compare immune cell activation and tumor cell killing between control and ZNF544-depleted cancer cells.

  • For macrophage experiments, assess phagocytosis rates using pH-sensitive fluorescent dyes.

  • For T cell experiments, measure activation markers, proliferation, and cytotoxic activity.

• Immunopeptidome analysis:

  • Immunoprecipitate HLA-I complexes from control vs. ZNF544-depleted cells.

  • Perform mass spectrometry to identify presented peptides.

  • Focus analysis on peptides derived from transposable elements or endogenous retroviruses.

• In vivo model considerations:

  • Establish syngeneic mouse models with ZNF544-depleted or overexpressing tumors.

  • Monitor tumor growth rates and immune infiltration.

  • Test responsiveness to immune checkpoint inhibitors.

  • Analyze tumor microenvironment changes through multiplexed immunohistochemistry.

These methodological approaches provide a framework for investigating how ZNF544 might contribute to immune evasion in cancer, potentially opening new avenues for therapeutic intervention.