ZNF564 Antibody

What is ZNF564 and what biological functions does it serve?

ZNF564 is a zinc finger protein that belongs to a family of proteins with versatile functions in eukaryotic genomes. Zinc finger proteins are one of the most abundant proteins in eukaryotes and are involved in critical biological processes including cell proliferation and apoptosis . Recent research has specifically implicated ZNF564 in neurological contexts, particularly in protection against bupivacaine-induced neurotoxicity . The protein functions as part of the regulatory ZFAS1/miR-421/ZNF564 axis, where it appears to play a protective role against neuronal damage . As a zinc finger protein, it likely functions in transcriptional regulation, though specific target genes and detailed mechanisms remain areas of active research.

What applications are ZNF564 antibodies suitable for in research?

ZNF564 antibodies are applicable across multiple research techniques, with validated applications including:

• Western Blotting (WB): Typically used at dilutions of 0.25 μg/mL in 5% skim milk/PBS buffer

• Immunofluorescence (IF): Recommended concentrations of 0.25-2 μg/mL

• Immunohistochemistry (IHC): Suggested dilutions of 1:50-1:200

• ELISA: Can be used at high dilutions (up to 1:312500 for peptide-based assays)

These applications make ZNF564 antibodies valuable tools for protein detection, localization studies, and quantitative analyses in diverse experimental settings.

What species reactivity can be expected with commercial ZNF564 antibodies?

ZNF564 is highly conserved across species, allowing for cross-reactivity of many commercial antibodies. Based on sequence homology analysis:

How can ZNF564 antibodies be used to investigate the ZFAS1/miR-421/ZNF564 regulatory axis in neurotoxicity studies?

The ZFAS1/miR-421/ZNF564 axis represents a complex regulatory network with significant implications for neurotoxicity research. To effectively investigate this pathway:

• Protein expression analysis: Use ZNF564 antibodies in Western blot analyses to quantify protein levels under various experimental conditions (e.g., bupivacaine treatment at different concentrations and time points) .

• Co-immunoprecipitation experiments: Combine ZNF564 antibodies with antibodies against ZFAS1-associated proteins to study physical interactions within the regulatory complex.

• Chromatin immunoprecipitation (ChIP): Apply ZNF564 antibodies in ChIP experiments to identify genomic binding sites and target genes.

• Cellular localization: Implement immunofluorescence with ZNF564 antibodies to track subcellular localization changes in response to pathway modulation .

• Pathway validation: Use the antibody in conjunction with miR-421 inhibitors or ZFAS1 overexpression/knockdown to confirm the regulatory relationship between these elements.

Research has demonstrated that ZNF564 expression is regulated by miR-421, which is itself sequestered by ZFAS1 acting as a competing endogenous RNA (ceRNA) . This regulatory axis has been shown to influence neuronal survival in bupivacaine-induced toxicity models, suggesting potential therapeutic applications for anesthetic-related neurotoxicity .

What methodological considerations are important when using ZNF564 antibodies for analyzing bupivacaine-induced neurotoxicity?

When investigating bupivacaine-induced neurotoxicity with ZNF564 antibodies, researchers should consider:

• Dose and time-dependent effects: ZNF564 expression changes in a manner dependent on both bupivacaine concentration and exposure duration. Experimental designs should incorporate multiple concentrations (0-2 mM range) and time points (0-48 hours) to capture the full response profile .

• Cell model selection: SH-SY5Y neuroblastoma cells have been validated for ZNF564 studies in neurotoxicity research, but additional cell lines should be considered to strengthen findings .

• Complementary molecular techniques: Combine protein detection (using ZNF564 antibodies) with mRNA quantification (RT-qPCR) to distinguish between transcriptional and post-transcriptional regulation .

• Functional assays: Correlate ZNF564 expression with functional outcomes such as:

  • Cell viability (MTT assay)

  • Apoptosis (TUNEL assay)

  • Caspase-3 activity measurements

• Genetic manipulation controls: Include appropriate controls when manipulating ZNF564 expression:

  • Overexpression controls (empty vector)

  • Knockdown controls (non-targeting shRNA)

  • Rescue experiments to confirm specificity

These methodological considerations ensure robust and reproducible results when studying the role of ZNF564 in neurotoxicity models.

How does ZNF564 interact with the miR-421 regulatory pathway at the molecular level?

The interaction between ZNF564 and miR-421 represents a classic miRNA-mediated regulatory mechanism:

• Direct targeting mechanism: miR-421 directly targets ZNF564 mRNA through complementary binding to specific sequences, as confirmed by luciferase reporter assays. When miR-421 binds to wild-type ZNF564 mRNA constructs, luciferase activity decreases significantly, while no effect is observed with mutated binding site constructs .

• Expression relationship: An inverse correlation exists between miR-421 and ZNF564 expression levels:

  • miR-421 overexpression leads to decreased ZNF564 expression

  • miR-421 inhibition results in increased ZNF564 expression

• Pathway regulation: The long non-coding RNA ZFAS1 acts as a molecular sponge for miR-421, preventing it from binding to ZNF564 mRNA, thus forming a complete regulatory circuit:

  • ZFAS1 ↑ → miR-421 availability ↓ → ZNF564 expression ↑

  • ZFAS1 ↓ → miR-421 availability ↑ → ZNF564 expression ↓

• Functional consequences: This regulatory relationship has direct implications for neuronal survival:

  • ZNF564 upregulation protects against bupivacaine-induced apoptosis

  • ZNF564 downregulation (via miR-421 upregulation) enhances neurotoxicity

Researchers can visualize these interactions using ZNF564 antibodies in combination with RNA immunoprecipitation (RIP) assays to capture the physical association between the protein and regulatory RNAs in the pathway.

What are the optimal conditions for using ZNF564 antibodies in Western blotting experiments?

For optimal Western blot results with ZNF564 antibodies:

• Sample preparation:

  • Lyse cells in standard RIPA buffer with protease inhibitors

  • Load 20-50 μg of total protein per lane

  • Use fresh samples when possible; avoid multiple freeze-thaw cycles

• Antibody dilution:

  • Primary antibody (ZNF564): 0.25 μg/mL in 5% skim milk/PBS buffer

  • Secondary antibody (HRP-conjugated anti-rabbit IgG): 1:50,000-1:100,000 dilution

• Blocking conditions:

  • 5% skim milk in PBS for 1 hour at room temperature

  • Alternative: 3% BSA in TBST for reduced background

• Incubation parameters:

  • Primary antibody: Overnight at 4°C with gentle rocking

  • Secondary antibody: 1 hour at room temperature

• Detection system:

  • Enhanced chemiluminescence (ECL) for standard applications

  • Fluorescence-based detection for quantitative analysis

Following these optimized conditions will help ensure specific detection of ZNF564 protein with minimal background interference.

How can ZNF564 antibodies be validated for specificity and cross-reactivity in neuroscience research?

Thorough validation of ZNF564 antibodies is essential for reliable research outcomes:

• Positive and negative controls:

  • Positive control: Cells/tissues known to express ZNF564 (e.g., SH-SY5Y cells)

  • Negative control: ZNF564 knockdown cells using siRNA or shRNA

  • Peptide competition: Pre-incubation with immunogenic peptide should abolish specific signal

• Cross-reactivity assessment:

  • Test antibody against recombinant ZNF564 from multiple species

  • Perform Western blots on lysates from various cell lines/tissues

  • Some commercial ZNF564 antibodies have been tested on protein arrays of 364 human recombinant protein fragments to confirm specificity

• Application-specific validation:

  • For IHC: Test on tissue microarrays containing multiple normal and cancer tissues (some antibodies have been validated on arrays of 44 normal human tissues and 20 common cancer types)

  • For IF: Confirm colocalization with other markers of expected subcellular compartments

• Batch-to-batch consistency:

  • Request Certificate of Analysis (CoA) for each antibody lot

  • Perform comparative testing between lots when possible

• Orthogonal methods:

  • Confirm protein expression using multiple antibodies targeting different epitopes

  • Correlate antibody-based detection with mRNA expression data

These validation steps ensure that experimental observations genuinely reflect ZNF564 biology rather than artifactual or non-specific signals.

What experimental design is recommended for studying ZNF564's role in the ZFAS1/miR-421/ZNF564 regulatory axis?

A comprehensive experimental design for investigating the ZFAS1/miR-421/ZNF564 axis should include:

• Expression profiling:

  • Measure baseline ZFAS1, miR-421, and ZNF564 levels in relevant cell models

  • Assess expression changes in response to bupivacaine treatment using:

    • RT-qPCR for RNA components

    • Western blot with ZNF564 antibodies for protein expression

• Pathway manipulation experiments:

  • Overexpression studies:

    • ZFAS1 overexpression → expected to increase ZNF564 levels

    • miR-421 overexpression → expected to decrease ZNF564 levels

    • ZNF564 overexpression → expected to provide neuroprotection

  • Knockdown studies:

    • ZFAS1 knockdown → expected to decrease ZNF564 levels

    • miR-421 inhibition → expected to increase ZNF564 levels

    • ZNF564 knockdown → expected to enhance neurotoxicity

• Rescue experiments:

  • ZFAS1 knockdown + miR-421 inhibitor → should restore ZNF564 levels

  • miR-421 overexpression + ZNF564 overexpression → should restore neuroprotection

• Mechanistic studies:

  • Luciferase reporter assays to confirm direct miR-421 binding to ZNF564

  • RNA immunoprecipitation to demonstrate physical interactions

  • ChIP-seq with ZNF564 antibodies to identify downstream targets

• Functional outcomes:

  • Cell viability assays (MTT)

  • Apoptosis detection (TUNEL assay)

  • Caspase-3 activity measurement

This systematic approach allows for comprehensive characterization of the regulatory relationships and functional consequences within the ZFAS1/miR-421/ZNF564 axis.

What are common challenges when using ZNF564 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with ZNF564 antibodies:

• Weak or absent signal:

  • Potential causes: Low expression levels, inefficient protein transfer, antibody degradation

  • Solutions: Increase protein loading, optimize transfer conditions, use fresh antibody aliquots

• Multiple bands in Western blot:

  • Potential causes: Protein isoforms, post-translational modifications, degradation products

  • Solutions: Use positive controls, optimize lysis conditions, add protease inhibitors

• High background in immunostaining:

  • Potential causes: Non-specific binding, inadequate blocking, excessive antibody concentration

  • Solutions: Increase blocking time, optimize antibody dilution, include additional washing steps

• Inconsistent results between experiments:

  • Potential causes: Antibody lot variation, sample preparation differences, protocol inconsistencies

  • Solutions: Standardize protocols, maintain detailed records, check for lot-to-lot consistency

• Cross-reactivity with related proteins:

  • Potential causes: Conserved domains within zinc finger protein family

  • Solutions: Use antibodies targeting unique epitopes, validate with knockdown controls

Proper storage is also critical - ZNF564 antibodies should be stored according to manufacturer recommendations (typically at -20°C with 50% glycerol to prevent freeze-thaw damage) .

How can researchers interpret conflicting data between ZNF564 protein levels and mRNA expression?

Discrepancies between ZNF564 protein and mRNA levels may arise from several biological mechanisms:

• Post-transcriptional regulation:

  • miRNAs (particularly miR-421) can suppress translation without affecting mRNA stability

  • RNA-binding proteins may alter translation efficiency

  • Solution: Measure both mRNA (RT-qPCR) and protein (Western blot with ZNF564 antibodies) to capture the complete regulatory picture

• Protein stability differences:

  • Post-translational modifications may affect protein half-life

  • Proteasomal degradation rates may vary under different conditions

  • Solution: Perform protein stability assays using cycloheximide chase experiments

• Temporal dynamics:

  • mRNA changes often precede protein level alterations

  • Solution: Conduct time-course experiments to capture the relationship between transcriptional and translational changes

• Compartmentalization effects:

  • Nuclear/cytoplasmic fractionation may affect apparent protein levels

  • Solution: Perform subcellular fractionation followed by Western blot analysis

• Technical considerations:

  • Different sensitivities between RNA and protein detection methods

  • Solution: Use quantitative methods for both mRNA (qPCR) and protein (quantitative Western blot)

When interpreting conflicting data, researchers should consider that the ZFAS1/miR-421/ZNF564 regulatory axis specifically operates through post-transcriptional mechanisms, where miR-421 regulates ZNF564 expression at the translational level .

What considerations are important when comparing ZNF564 antibody results across different experimental models?

When comparing ZNF564 antibody results across different experimental systems:

• Species-specific differences:

  • Despite high conservation (81-100% identity across species), subtle sequence variations may affect antibody binding

  • Solution: Use antibodies validated for the specific species under investigation, or confirm cross-reactivity experimentally

• Cell type-specific expression patterns:

  • ZNF564 expression levels may vary dramatically between different cell types

  • Solution: Include positive control samples from relevant tissues/cell lines in each experiment

• Experimental condition standardization:

  • Variations in culture conditions, treatment protocols, or sample preparation can affect results

  • Solution: Maintain consistent protocols across experiments and clearly document any variations

• Antibody selection:

  • Different antibodies may target distinct epitopes, leading to apparent discrepancies

  • Solution: Use multiple antibodies targeting different regions of ZNF564 when possible

• Data normalization approaches:

  • Various normalization strategies (housekeeping proteins, total protein staining) may influence comparative analyses

  • Solution: Apply consistent normalization methods across all experimental models

• Validation in multiple systems:

  • Findings in cell lines (e.g., SH-SY5Y) may not translate directly to primary neurons or in vivo models

  • Solution: Confirm key findings in at least two independent model systems

Current research on ZNF564 in neurotoxicity has acknowledged limitations in model diversity, noting the need for verification beyond SH-SY5Y cells in future studies .

What are promising research areas for ZNF564 antibody applications beyond neurotoxicity studies?

While current research has focused on ZNF564's role in neurotoxicity, several promising directions for future investigation include:

• Broader neurological disease contexts:

  • Parkinson's Disease: Given that miR-421 facilitates neuronal apoptosis in PD, and ZNF564 is regulated by miR-421, investigating ZNF564's role in PD pathogenesis is warranted

  • Epilepsy: miR-421 has established roles in epilepsy, suggesting potential involvement of the ZFAS1/miR-421/ZNF564 axis

  • Alzheimer's Disease: Other lncRNAs like NEAT1 affect neuronal injury in AD, suggesting similar mechanisms might involve ZNF564

• Cancer biology:

  • The ZFAS1/miR-421 interaction has been documented in oral squamous cell carcinoma, suggesting potential roles for ZNF564 in cancer contexts

  • ZNF564 antibodies could be valuable for investigating its expression patterns across diverse tumor types

• Developmental biology:

  • The high conservation of ZNF564 across species (from humans to zebrafish) suggests important evolutionary functions

  • ZNF564 antibodies could help map expression patterns during developmental processes

• Transcriptional regulation:

  • As a zinc finger protein, ZNF564 likely functions in transcriptional regulation

  • ChIP-seq studies using ZNF564 antibodies could identify genomic binding sites and target genes

• Therapeutic target validation:

  • If ZNF564 proves protective in multiple neurological contexts, antibody-based detection methods will be essential for validating and monitoring therapeutic interventions targeting this pathway

Each of these research directions would benefit from the application of well-validated ZNF564 antibodies for protein detection, localization, and functional studies.