ZNF554 Antibody

What is ZNF554 and why is it important in neurological research?

ZNF554 (Zinc finger protein 554) is a member of the Krüppel-associated box domain zinc finger protein subfamily that shows predominant expression in the brain with low region specificity in humans. It has emerged as an important research target due to its potential role as a tumor suppressor in gliomas and other brain cancers. The protein demonstrates decreased expression with increasing glioma grades, and lower expression is associated with worse prognosis in grade III astrocytomas and glioblastomas . When studying neurological disorders and brain tumors, ZNF554 antibodies allow researchers to detect and quantify this protein across different brain regions and pathological states, providing insights into disease mechanisms and potential therapeutic targets.

What are the recommended methods for validating ZNF554 antibody specificity?

When validating ZNF554 antibody specificity, researchers should employ multiple complementary approaches:

• Western blotting with positive controls (brain tissue) and negative controls (tissues with minimal ZNF554 expression)

• Immunohistochemistry (IHC) on brain tissue sections with appropriate blocking controls

• Competitive inhibition assays using recombinant ZNF554 protein

• Testing in ZNF554 knockout or knockdown models

• Immunoprecipitation followed by mass spectrometry to confirm target pull-down

Validation is particularly important when studying ZNF554 in gliomas, where expression levels vary significantly with tumor grade . A properly validated antibody should show decreased immunostaining intensity that correlates with higher glioma grades, consistent with the transcriptomic data indicating reduced ZNF554 expression in higher-grade tumors.

How can ZNF554 antibodies be optimized for improved stability and affinity in brain tissue research?

Optimization of ZNF554 antibodies for brain tissue research can be approached using computational and experimental methods:

• Computational optimization: Deep learning models like DeepAb can be employed to predict antibody Fv structure from sequence data and identify potentially stabilizing mutations . For ZNF554 antibodies, this approach can help design variants with enhanced thermal and colloidal stability without compromising target specificity.

• Experimental validation workflow:

  • Generate 200+ variants through high-throughput methods

  • Test for thermal stability (Tonset, Tm, Tagg)

  • Evaluate binding affinity (KD) compared to parental antibody

  • Assess developability parameters (non-specific binding, aggregation propensity)

In one study using DeepAb for antibody optimization, 91% of designed variants showed increased thermal stability and 94% demonstrated enhanced binding affinity . While this methodology wasn't specifically applied to ZNF554 antibodies, it provides a framework that can be adapted for optimizing antibodies targeting this protein in brain tissue research.

What are the methodological considerations when using ZNF554 antibodies for quantifying expression differences between normal brain and glioma tissues?

When quantifying ZNF554 expression differences between normal and glioma tissues using antibodies, several methodological considerations are critical:

• Tissue preparation standardization:

  • Use consistent fixation protocols (preferably 4% paraformaldehyde)

  • Standardize antigen retrieval methods (heat-induced epitope retrieval at pH 6.0 is often optimal)

  • Include positive controls (normal brain) and negative controls (tissues with minimal ZNF554 expression)

• Quantification approaches:

  • Use semi-quantitative immunoscoring (0-3 scale) as employed in previous studies

  • Implement digital image analysis with appropriate software for more objective quantification

  • Normalize staining intensity to internal controls

• Data interpretation challenges:

  • Account for heterogeneity within tumors by analyzing multiple regions

  • Consider IDH mutation status when interpreting ZNF554 expression in gliomas

  • Correlate immunohistochemistry findings with mRNA expression data when possible

Research has shown that ZNF554 immunoscores are 33% and 47% lower in grade II and grade III oligodendrogliomas respectively, and 40% and 52% lower in grade III astrocytomas and glioblastomas respectively, compared to controls . These substantial differences highlight the importance of precise quantification methods.

How can researchers address potential discrepancies between ZNF554 mRNA and protein expression levels in experimental models?

Discrepancies between ZNF554 mRNA and protein expression levels present a common challenge in research. To address this issue:

• Integrated measurement approach:

  • Perform parallel qRT-PCR and Western blotting/IHC analyses

  • Include appropriate housekeeping genes and proteins for normalization

  • Analyze samples at multiple time points to capture dynamic expression changes

• Investigating regulatory mechanisms:

  • Assess microRNA regulation of ZNF554 mRNA translation

  • Examine protein degradation pathways using proteasome inhibitors

  • Evaluate post-translational modifications that may affect antibody recognition

• Validation strategies:

  • Utilize multiple antibodies targeting different ZNF554 epitopes

  • Implement CRISPR-Cas9 ZNF554 knockout controls

  • Employ polysome profiling to assess translational efficiency

Previous research on ZNF554 in gliomas demonstrated consistency between mRNA and protein expression patterns across different tumor grades , but this may not hold true in all experimental conditions or model systems, necessitating careful validation.

What is the significance of ZNF554 as a prognostic marker in gliomas, and how can antibodies help in patient stratification?

ZNF554 has emerged as a potential prognostic marker in gliomas, with significant implications for patient stratification:

Analysis of TCGA RNA-Seq data (n=687) demonstrated that patients with high ZNF554 expression have significantly better survival probability compared to those with low expression (p<0.001) . This finding suggests that antibody-based detection of ZNF554 could become an important component of glioma prognostic assessment.

How can researchers design ZNF554 antibody-based assays to investigate its interaction with the PI3K-Akt signaling pathway in glioblastoma?

Designing ZNF554 antibody-based assays to investigate interactions with the PI3K-Akt pathway requires careful consideration:

• Co-immunoprecipitation approaches:

  • Use ZNF554 antibodies conjugated to solid supports (magnetic beads/agarose)

  • Lyse glioblastoma cells under conditions that preserve protein complexes

  • Probe precipitates for PI3K-Akt pathway components (p85, p110, AKT, mTOR)

  • Include appropriate controls (IgG precipitation, reciprocal co-IP)

• Proximity ligation assays (PLA):

  • Employ paired antibodies against ZNF554 and key PI3K-Akt components

  • Visualize and quantify protein interactions in situ within glioblastoma tissue sections

  • Compare interaction patterns between different tumor grades

• Functional assays following manipulation:

  • Overexpress ZNF554 in glioblastoma cell lines as demonstrated previously

  • Monitor changes in PI3K-Akt pathway activation (phosphorylation states)

  • Combine with PI3K-Akt inhibitors to assess pathway interdependence

This approach is supported by previous findings showing that the "PI3K-Akt signaling pathway" was the most impacted among 116 dysregulated pathways when ZNF554 was overexpressed in U87 glioblastoma cells .

What considerations should be made when designing experimental controls for ZNF554 antibody-based assays in glioma research?

Proper experimental controls are crucial for ZNF554 antibody-based assays in glioma research:

• Positive and negative tissue controls:

  • Normal brain tissue (high ZNF554 expression) as positive control

  • Non-neural tissues with minimal ZNF554 expression as negative controls

  • Glioma tissues of different grades to demonstrate gradation of expression

• Genetic controls:

  • CRISPR-Cas9 knockout of ZNF554 in glioma cell lines

  • siRNA knockdown samples with validated reduction in ZNF554 expression

  • ZNF554 overexpression models as demonstrated in U87 cells

• Antibody validation controls:

  • Secondary antibody-only controls to assess non-specific binding

  • Isotype controls matched to the ZNF554 antibody

  • Peptide competition/absorption controls using recombinant ZNF554

• Experimental design controls:

  • Include samples from recurrent and primary gliomas (shown to have similar ZNF554 expression)

  • Stratify samples by IDH mutation status

  • Normalize to multiple reference genes/proteins

Properly controlled experiments are essential since ZNF554 expression shows subtle but significant differences across glioma grades, with immunoscores decreasing progressively from normal brain to higher-grade tumors .

How can ChIP-seq with ZNF554 antibodies be optimized to identify its gene targets in normal brain versus glioma tissues?

Optimizing ChIP-seq with ZNF554 antibodies requires several technical considerations:

• Antibody qualification for ChIP applications:

  • Validate antibody specificity using Western blotting and immunoprecipitation

  • Perform pilot ChIP-qPCR on known/predicted ZNF554 binding sites

  • Test multiple antibody lots and concentrations to determine optimal conditions

• Tissue-specific protocol adaptations:

  • For brain tissue: Implement crosslinking conditions that account for high lipid content

  • For glioma samples: Adjust cell lysis and sonication parameters based on tumor grade

  • Consider using CUT&RUN or CUT&Tag as alternatives for limited clinical samples

• Data analysis approach:

  • Use appropriate peak calling algorithms (MACS2 with input normalization)

  • Perform differential binding analysis between normal brain and glioma samples

  • Correlate binding sites with genes dysregulated in ZNF554-overexpressing glioblastoma cells

Previous research identified approximately 150 gene promoters as binding sites for ZNF554 in the brain . ChIP-seq could expand this knowledge by characterizing how these binding patterns change in gliomas where ZNF554 expression is decreased.

What methodological approaches can resolve contradictory data between ZNF554 expression and function in different glioma subtypes?

Resolving contradictory data regarding ZNF554 expression and function across glioma subtypes requires systematic approaches:

• Integrated multi-omics analysis:

  • Correlate ZNF554 protein levels (antibody-based detection) with mRNA expression

  • Perform parallel DNA methylation analysis of the ZNF554 promoter

  • Assess chromatin accessibility at the ZNF554 locus using ATAC-seq

• Subtype-specific functional studies:

  • Generate isogenic glioma cell lines representing different molecular subtypes

  • Implement identical ZNF554 overexpression/knockdown protocols across all models

  • Measure consistent functional readouts (proliferation, invasion, cell cycle)

• Patient-derived models:

  • Establish patient-derived xenografts from different glioma subtypes

  • Evaluate ZNF554 expression and response to manipulation in these models

  • Correlate findings with original patient tumor characteristics

Research has shown variable survival advantages of high ZNF554 expression across different glioma subtypes, with significant effects in grade III astrocytomas and glioblastomas but not in oligodendrogliomas or low-grade astrocytomas . These differences warrant detailed investigation using the approaches outlined above.

How can biolayer interferometry be optimized for characterizing ZNF554 antibody binding kinetics and affinity?

Biolayer interferometry (BLI) optimization for ZNF554 antibody characterization requires attention to several technical parameters:

• Sensor selection and loading strategy:

  • Use anti-human FAB2G Octet probes for consistent antibody orientation

  • Optimize antibody loading concentration to achieve 0.8-1.2 nm shift

  • Implement slow loading rates (30-50 µg/mL) to minimize avidity effects

• Binding kinetics protocol design:

  • Prepare ZNF554 antigen in 5-7 concentrations spanning 0.1-10× KD

  • Include zero-concentration controls to correct for baseline drift

  • Set association times sufficient to approach equilibrium (typically 300-600 seconds)

  • Allow full dissociation (600-1200 seconds) for accurate koff determination

• Data analysis considerations:

  • Apply global fitting to the entire concentration series

  • Compare 1:1 binding models with more complex models (heterogeneous ligand)

  • Validate BLI results with orthogonal methods like surface plasmon resonance (SPR)

This approach is informed by antibody-antigen interaction studies that demonstrated consistency between BLI and SPR measurements, where one antibody showed KD values of 6 nM (IgG) and 3.75 nM (Fab) by BLI, and 0.93 nM (IgG) and 1.19 nM (Fab) by SPR .

What experimental design is recommended for investigating the role of ZNF554 in cell cycle regulation using antibody-based detection methods?

For investigating ZNF554's role in cell cycle regulation, the following experimental design is recommended:

• Cell synchronization and analysis protocol:

  • Synchronize glioblastoma cells at different cell cycle phases using standard methods

  • Harvest cells at defined time points for ZNF554 immunoblotting

  • Perform parallel flow cytometry with propidium iodide staining for cell cycle validation

  • Quantify ZNF554 levels relative to housekeeping proteins at each time point

• ZNF554 manipulation experiments:

  • Establish stable cell lines with inducible ZNF554 overexpression

  • Induce ZNF554 at different cell cycle phases

  • Measure G0/G1 arrest as previously observed in U87 cells

  • Quantify expression of cell cycle regulators affected by ZNF554 manipulation

• Immunofluorescence co-localization studies:

  • Perform double immunostaining for ZNF554 and cell cycle markers

  • Use high-resolution confocal microscopy to determine subcellular localization

  • Quantify co-localization coefficients at different cell cycle stages

This approach builds on previous observations that ZNF554 overexpression in U87 glioblastoma cells led to increased cells in G0/G1 phase (from 46.65% to 48.05%) , suggesting cell cycle arrest as a mechanism of proliferation inhibition.

How should researchers design sandwich ELISA protocols for quantifying ZNF554 in clinical glioma specimens?

Designing sandwich ELISA protocols for ZNF554 quantification in clinical specimens requires careful optimization:

• Antibody pair selection strategy:

  • Screen multiple monoclonal antibodies recognizing distinct ZNF554 epitopes

  • Evaluate capture/detection antibody combinations for optimal signal-to-noise ratio

  • Validate antibody performance against recombinant ZNF554 standards

• Sample preparation protocol:

  • Optimize tissue homogenization buffer composition (consider detergent types/concentrations)

  • Determine ideal protein concentration range (typically 0.1-1.0 mg/mL total protein)

  • Establish consistent freeze-thaw limits to maintain ZNF554 integrity

• Assay validation requirements:

  • Generate standard curves using recombinant ZNF554 (0.1-100 ng/mL range)

  • Determine lower limit of detection and quantification

  • Assess intra-assay and inter-assay variability (target CV <15%)

  • Perform spike-recovery experiments to evaluate matrix effects

• Clinical application parameters:

  • Determine reference ranges for normal brain and different glioma grades

  • Establish clinically relevant cutoff values based on survival correlations

  • Implement quality control samples across multi-center studies

This approach would allow for quantitative comparison of ZNF554 across glioma grades, potentially complementing the semi-quantitative immunohistochemistry approaches previously used to demonstrate decreased ZNF554 with increasing tumor grade .

What methodological approaches can determine how ZNF554 regulates the expression of cytokines and chemokines in glioblastoma?

To investigate ZNF554's regulation of cytokines and chemokines in glioblastoma, researchers should consider these methodological approaches:

• Gene regulation analysis workflow:

  • Perform ZNF554 ChIP-seq to identify direct binding to cytokine/chemokine promoters

  • Conduct luciferase reporter assays with wild-type and mutated promoter regions

  • Implement CRISPR activation/interference targeted to ZNF554 binding sites

• Protein expression analysis:

  • Quantify cytokine/chemokine levels using multiplex immunoassays following ZNF554 manipulation

  • Compare secreted vs. intracellular levels to assess post-transcriptional regulation

  • Validate findings using targeted Western blotting with specific antibodies

• Functional validation experiments:

  • Conduct rescue experiments using recombinant cytokines/chemokines in ZNF554-overexpressing cells

  • Implement cytokine/chemokine receptor blocking to assess pathway dependence

  • Evaluate effects on immune cell recruitment using transwell migration assays

Previous research showed that among the top 20 upregulated genes in ZNF554-overexpressing glioblastoma cells were several cytokines (IL6, IL18, IL36B) and chemokines (CCL20, CCL5, CXCL2) , suggesting a direct regulatory relationship that warrants detailed mechanistic investigation.

How can researchers use ZNF554 antibodies to investigate its potential role in immune regulation within the glioma microenvironment?

Investigating ZNF554's role in immune regulation within the glioma microenvironment requires strategic use of antibodies:

• Multiplex immunohistochemistry approach:

  • Perform co-staining of ZNF554 with immune cell markers (CD4, CD8, CD68, etc.)

  • Quantify spatial relationships between ZNF554-expressing cells and immune infiltrates

  • Compare patterns across glioma grades and correlation with patient outcomes

• Ex vivo tumor slice culture methodology:

  • Maintain fresh glioma tissue slices in culture with preserved microenvironment

  • Treat with anti-ZNF554 neutralizing antibodies or ZNF554 overexpression

  • Monitor changes in immune cell composition and activation status

• Single-cell analysis protocol:

  • Perform single-cell RNA-seq on glioma samples with varying ZNF554 expression

  • Use antibodies to sort ZNF554-high versus ZNF554-low cell populations

  • Correlate ZNF554 levels with immune pathway activation signatures

This approach is supported by findings that 86 out of 116 dysregulated pathways in ZNF554-overexpressing glioblastoma cells were immune-related , suggesting ZNF554 may play a significant role in modulating immune responses in the glioma microenvironment.