ZNF644 Antibody

What is ZNF644 and why is it important in molecular biology research?

ZNF644 (Zinc Finger Protein 644) functions as a transcription factor critically involved in gene regulation, cell differentiation, and development. It serves as a core subunit in the G9a/GLP complex, which mediates mono- and dimethylation of lysine 9 on histone H3 (H3K9) at specific gene loci . This epigenetic modification is strongly associated with transcriptional repression mechanisms. The importance of ZNF644 in research stems from its fundamental role in controlling gene expression patterns and cell fate decisions, making it a significant target for studies in molecular biology, genetics, and disease mechanisms . Research has demonstrated that ZNF644 contains multiple zinc finger motifs that recognize specific DNA sequences, enabling it to target the G9a/GLP complex to particular genomic regions for H3K9 methylation and subsequent gene silencing .

What are the primary applications for ZNF644 antibodies in experimental research?

ZNF644 antibodies serve multiple critical applications in molecular and cellular research:

• Western Blotting (WB): For detecting and quantifying ZNF644 protein expression levels in cell or tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For measuring ZNF644 protein concentrations in solution with high sensitivity

• Immunoprecipitation (IP): For isolating ZNF644 and its associated protein complexes

• Immunohistochemistry (IHC): For visualizing the cellular and tissue distribution of ZNF644

• Chromatin Immunoprecipitation (ChIP): For identifying genomic binding sites of ZNF644

The application versatility allows researchers to study ZNF644 at multiple experimental levels, from protein expression to functional genomics.

What species reactivity should researchers consider when selecting ZNF644 antibodies?

When selecting ZNF644 antibodies, researchers must carefully consider species reactivity based on their experimental model. Available antibodies demonstrate varied cross-reactivity profiles:

The extensive cross-reactivity of certain antibodies makes them valuable for comparative studies across species, while antibodies with narrower specificity may be preferred for human-focused research .

What are the optimal conditions for using ZNF644 antibodies in Western blotting?

For optimal Western blotting results with ZNF644 antibodies, researchers should consider these methodological parameters:

• Expected molecular weight: ZNF644 typically appears at approximately 150 kDa on SDS-PAGE gels

• Sample preparation: Cell lysates should be prepared using NETN400 buffer (0.5% NP-40, 50 mM Tris-HCl pH 8.0, 2 mM EDTA, and 400 mM NaCl) supplemented with phosphatase inhibitors (10 mM NaF and 50 mM β-glycerophosphate)

• Dilution: For ELISA applications, optimal dilution is approximately 1:62500

• Controls: Include siRNA-treated samples to validate antibody specificity, as demonstrated in previous studies where siZNF644 treatment diminished the expression of the targeted protein

• Detection method: Secondary antibodies conjugated to HRP or fluorophores can be used depending on the desired sensitivity and equipment availability

Optimization may be required for different cell types or experimental conditions to ensure specific detection of ZNF644.

How can researchers validate the specificity of ZNF644 antibodies?

Validating antibody specificity is critical for ensuring reliable experimental results. For ZNF644 antibodies, researchers should implement multiple validation strategies:

• RNA interference: Treating cells with siRNAs targeting ZNF644 should result in reduced antibody signal. Previous studies have confirmed the specificity of ZNF644 antibodies by demonstrating that siZNF644 treatment diminished the expression of the 150 kDa band recognized by the antibody .

• Immunogenic peptide competition: Pre-incubating the antibody with the immunogenic peptide used for antibody production should block specific binding.

• Recombinant protein controls: Using purified ZNF644 protein or overexpression systems as positive controls.

• Cross-validation with different antibodies: Using multiple antibodies targeting different epitopes of ZNF644 to confirm consistent results.

• Molecular weight verification: Confirming that the detected band appears at the expected molecular weight of approximately 150 kDa .

These validation steps collectively ensure that experimental observations genuinely reflect ZNF644 biology rather than non-specific interactions.

What approaches can be used to troubleshoot non-specific binding with ZNF644 antibodies?

When encountering non-specific binding with ZNF644 antibodies, researchers can implement these troubleshooting approaches:

• Optimize blocking conditions: Increase blocking time or try alternative blocking agents (BSA, non-fat milk, commercial blocking buffers) to reduce background.

• Adjust antibody concentration: Titrate the primary antibody to determine the optimal concentration that maximizes specific signal while minimizing background.

• Modify washing procedures: Increase the number or duration of washes, or adjust salt concentration in wash buffers. For ZNF644-related experiments, NETN buffer with varying salt concentrations (300-400 mM NaCl) has been successfully used .

• Pre-adsorb the antibody: Incubate the antibody with tissues or cells lacking ZNF644 expression to remove cross-reactive antibodies.

• Use purified antibodies: Antibodies purified by peptide affinity chromatography, as used in previous ZNF644 studies, generally show improved specificity .

• Employ additional controls: Include knockout/knockdown samples as negative controls to definitively identify specific versus non-specific bands.

How can ZNF644 antibodies be utilized to study protein-protein interactions in the G9a/GLP complex?

ZNF644 antibodies serve as powerful tools for investigating protein-protein interactions within the G9a/GLP complex through these advanced approaches:

• Co-immunoprecipitation (Co-IP): ZNF644 antibodies can be used to pull down the protein along with its interacting partners. Previous research successfully employed this technique to demonstrate that ZNF644 associates with other core components of the G9a/GLP complex, including WIZ, G9a, and GLP .

• Proximity ligation assay (PLA): This technique can visualize protein-protein interactions in situ when using ZNF644 antibodies in combination with antibodies against suspected interaction partners.

• ChIP-reChIP: Sequential chromatin immunoprecipitation can determine if ZNF644 and other G9a/GLP complex components co-occupy the same genomic regions.

• Immunofluorescence co-localization: ZNF644 antibodies paired with antibodies against other complex components can reveal spatial relationships within nuclear compartments.

• Pull-down assays with recombinant domains: When combined with domain mapping strategies, ZNF644 antibodies can help determine which protein regions mediate specific interactions, as demonstrated in studies showing that ZNF644 interacts with the transcription activation domain of G9a .

These approaches collectively provide a comprehensive understanding of how ZNF644 functions within the larger epigenetic regulatory complex.

What role does ZNF644 play in epigenetic regulation and how can antibodies help elucidate this function?

ZNF644 serves a critical function in epigenetic regulation by targeting the G9a/GLP histone methyltransferase complex to specific genomic loci, thereby influencing chromatin structure and gene expression. ZNF644 antibodies can help elucidate these functions through several methodological approaches:

• Chromatin Immunoprecipitation (ChIP): ZNF644 antibodies enable the identification of genomic regions bound by ZNF644. When coupled with sequencing (ChIP-seq), this technique provides genome-wide binding profiles, revealing potential regulatory targets.

• Sequential ChIP experiments: These can determine co-occupancy of ZNF644 with H3K9me2 marks and other G9a/GLP complex components at specific genomic regions.

• Functional correlation studies: By combining ZNF644 ChIP data with transcriptome analysis following ZNF644 depletion, researchers can identify genes directly regulated by ZNF644-mediated H3K9 methylation. Previous research has demonstrated that ZNF644 and WIZ target the G9a/GLP complex to genomic loci for H3K9 methylation and transcriptional repression .

• Mechanistic studies: ZNF644 antibodies can help investigate how the zinc finger motifs in ZNF644 recognize specific DNA sequences, a crucial aspect of its targeting function .

• Analysis of coordinated gene repression: Recent research indicates that genes upregulated following depletion of EHMT2 (G9a) form clusters along the linear genome, suggesting coordinated repression mechanisms that may involve ZNF644 .

Understanding ZNF644's epigenetic functions has broad implications for developmental biology and disease pathogenesis.

How can researchers design experiments to study the DNA binding specificity of ZNF644?

To investigate the DNA binding specificity of ZNF644, researchers can implement these experimental approaches:

• Electrophoretic Mobility Shift Assay (EMSA): This technique can determine if ZNF644 directly binds to specific DNA sequences. Previous studies have successfully used this approach with GST-tagged ZNF644 proteins and radio-labeled DNA substrates containing potential binding motifs . The experimental protocol included:

  • Incubating GST-ZNF644 with 0.2 nM radio-labeled DNA substrates for 2 hours at 4°C

  • Using buffer D (20 mM HEPES-KOH pH 7.9, 20% glycerol, 0.2 mM EDTA, 0.1 M KCl, 0.5 mM PSMF, 1 mM DTT)

  • Including BSA (1.25 μg/μl), DTT (1 mM), and MgCl₂ (5 mM)

  • Resolving samples by electrophoresis on a 7.5% polyacrylamide gel in TBE buffer

• ChIP-seq with motif analysis: ZNF644 antibodies can be used for ChIP-seq experiments, followed by computational motif discovery to identify consensus binding sequences.

• SELEX (Systematic Evolution of Ligands by Exponential Enrichment): This technique can identify high-affinity binding sequences for ZNF644 from random DNA libraries.

• Mutational analysis: Systematically testing the effect of mutations in putative binding sequences on ZNF644 binding, as demonstrated in previous research using wild-type and mutant DNA targets .

• Domain mapping: Using antibodies against different domains of ZNF644 to determine which zinc finger motifs are responsible for specific DNA interactions.

These approaches provide complementary information about the sequence specificity and binding characteristics of ZNF644.

What are the current challenges in studying ZNF644's role in the G9a/GLP complex?

Despite significant advances, several challenges persist in studying ZNF644's role in the G9a/GLP complex:

• Distinguishing direct from indirect effects: Determining which gene expression changes following ZNF644 perturbation are directly caused by altered targeting of the G9a/GLP complex versus secondary effects.

• Context-dependent functions: Understanding how ZNF644's function varies across different cell types, developmental stages, or disease states requires comprehensive analysis across multiple systems.

• Redundancy and compensation: Elucidating potential functional overlap between ZNF644 and other zinc finger proteins that might compensate for its loss.

• Technological limitations: Current antibodies may not distinguish between different ZNF644 isoforms or post-translationally modified forms, potentially masking important regulatory mechanisms.

• Temporal dynamics: Capturing the dynamic nature of ZNF644 binding and G9a/GLP complex assembly during cellular processes requires sophisticated time-course experiments.

• Integration with 3D genome organization: Understanding how ZNF644-mediated repression relates to higher-order chromatin structures and topologically associating domains (TADs), as suggested by recent research showing that DEGUP clusters can span across TAD boundaries .

Addressing these challenges requires interdisciplinary approaches combining genomics, proteomics, and advanced imaging techniques.

How can ZNF644 antibodies be used to investigate the molecular mechanisms of diseases linked to ZNF644 dysregulation?

ZNF644 dysregulation has been implicated in various diseases including cancer, neurological disorders, and developmental defects . ZNF644 antibodies can facilitate investigations into disease mechanisms through multiple approaches:

• Expression profiling: Quantifying ZNF644 protein levels in diseased versus normal tissues using Western blotting or immunohistochemistry with ZNF644 antibodies to identify expression patterns associated with pathological states.

• Epigenomic alterations: Combining ChIP-seq using ZNF644 antibodies with H3K9me2 profiling to map disease-specific changes in chromatin modification patterns.

• Interactome analysis: Using co-immunoprecipitation with ZNF644 antibodies followed by mass spectrometry to identify alterations in protein-protein interactions in disease contexts.

• Functional rescue experiments: Assessing whether restoring normal ZNF644 levels or function can reverse disease phenotypes in cellular or animal models.

• Target gene regulation: Investigating whether disease-associated mutations in ZNF644 alter its ability to regulate specific target genes by comparing ChIP and gene expression data between wild-type and mutant conditions.

• Cell-specific effects: Using ZNF644 antibodies for single-cell analysis techniques to understand cell type-specific roles in heterogeneous disease tissues.

These approaches collectively provide mechanistic insights into how ZNF644 dysfunction contributes to disease pathogenesis and may identify potential therapeutic targets.

What are the cutting-edge techniques for studying ZNF644-mediated transcriptional repression?

Research into ZNF644-mediated transcriptional repression continues to advance through innovative techniques:

• CUT&RUN and CUT&Tag: These techniques provide higher resolution and lower background compared to traditional ChIP-seq for mapping ZNF644 binding sites and associated histone modifications.

• HiChIP and PLAC-seq: These methods combine chromatin immunoprecipitation with chromosome conformation capture to understand how ZNF644 influences 3D genome organization and long-range gene regulation.

• CRISPR screening: CRISPR-based approaches can systematically disrupt ZNF644 binding sites or interacting partners to identify functional dependencies.

• Single-molecule imaging: Techniques like STORM or PALM can visualize the dynamics of ZNF644 and the G9a/GLP complex assembly at specific genomic loci in living cells.

• Proteomics approaches: Proximity labeling techniques (BioID, APEX) can identify proteins in close proximity to ZNF644 in different cellular compartments or conditions.

• Integrative genomic analysis: Computational approaches that integrate ZNF644 binding data with other epigenomic marks, gene expression, and 3D genome organization have revealed that many strongly upregulated genes following G9a/GLP complex disruption are organized into clusters along the linear genome .

These cutting-edge approaches provide unprecedented insights into the molecular mechanisms underlying ZNF644's role in transcriptional regulation.

What are the considerations for using ZNF644 antibodies in ChIP-seq experiments?

When designing ChIP-seq experiments with ZNF644 antibodies, researchers should consider these critical factors:

• Antibody selection: Choose antibodies validated specifically for ChIP applications. Polyclonal antibodies generated against larger regions of ZNF644 (such as N-terminus a.a. 50-602) have demonstrated success in immunoprecipitation experiments .

• Fixation conditions: Optimize formaldehyde concentration and cross-linking time for ZNF644, which as a transcription factor may require different conditions than histone proteins.

• Sonication parameters: Adjust sonication conditions to generate optimal DNA fragment sizes (typically 200-500 bp) while preserving epitope integrity.

• Controls:

  • Input DNA controls

  • IgG negative controls

  • Positive controls targeting known ZNF644-regulated regions

  • Biological replicates (minimum of 2-3)

  • Knockdown/knockout samples as additional specificity controls

• Sequential ChIP considerations: For co-occupancy studies with G9a/GLP or H3K9me2, optimize antibody combinations and wash conditions to maintain complex integrity.

• Data analysis pipeline: Employ appropriate peak calling algorithms and integrate with existing datasets on G9a/GLP binding and H3K9me2 distribution for comprehensive analysis.

• Validation: Confirm key findings with orthogonal techniques such as ChIP-qPCR targeting specific genomic regions of interest.

These considerations help ensure generation of high-quality, interpretable ChIP-seq data for understanding ZNF644's genomic targets.

How should researchers approach the analysis of contradictory data when studying ZNF644 function?

When encountering contradictory data in ZNF644 research, investigators should implement a systematic analysis approach:

This structured approach helps resolve apparent contradictions and develops a more nuanced understanding of ZNF644 biology.