ZNF331 Antibody

What is ZNF331 and what are its key structural characteristics?

ZNF331 (Zinc finger protein 331) belongs to the krueppel C2H2-type zinc-finger protein family. It contains 12 C2H2-type zinc fingers and 1 KRAB (Kruppel-related box) domain, which is commonly found in transcriptional repressors. The zinc finger domains are conserved amino acid sequence motifs containing 2 specifically positioned cysteines and 2 histidines that coordinate zinc atoms. ZNF331 has a calculated molecular weight of approximately 53.7 kDa and is encoded by a gene located on chromosome 19q13.42. The protein is also known by alternative names including RITA, ZNF361, ZNF463, and C2H2-like zinc finger protein rearranged in thyroid adenomas .

What are the validated applications for ZNF331 antibodies?

ZNF331 antibodies have been validated for multiple experimental applications, with validation varying by specific antibody product. Based on current research literature and commercial offerings, the following applications have been validated:

When selecting an antibody, researchers should verify that it has been validated for their specific application and species of interest, as validation varies across manufacturers .

What is the recommended storage and handling protocol for ZNF331 antibodies?

For optimal performance and longevity of ZNF331 antibodies, the following storage and handling guidelines are recommended:

• Store at -20°C for long-term storage (up to one year)

• For frequent use and short-term storage (up to one month), store at 4°C

• Avoid repeated freeze-thaw cycles as they can degrade antibody performance

• Most ZNF331 antibodies are supplied in liquid form containing buffer components such as PBS with 0.02% sodium azide and 50% glycerol at pH 7.2

• When working with the antibody, keep it on ice when not in storage

• Follow manufacturer-specific recommendations, as formulations may vary slightly between suppliers

How does ZNF331 expression differ between normal and pathological tissue samples?

Research has revealed significant differences in ZNF331 expression between normal and pathological tissues, particularly in glioma. Expression analysis studies have demonstrated:

• ZNF331 is significantly reduced in low-grade glioma tissue (WHO I-II) compared to normal brain tissue

• Expression is further reduced in high-grade glioma tissue (WHO III-IV) compared to low-grade glioma tissues

• In U87 and U251 glioma cell lines, ZNF331 expression levels are significantly lower than in normal human astrocytes (NHA)

• This progressive reduction in expression correlates with increasing malignancy grade

These findings suggest ZNF331 may function as a tumor suppressor in glioma. Similar patterns of reduced expression have been documented in other cancers, including colorectal cancer where low ZNF331 expression is associated with poor prognosis, and gastric cancer where promoter methylation leads to ZNF331 inactivation .

What experimental controls should be included when validating ZNF331 antibody specificity?

To ensure robust and reproducible results when working with ZNF331 antibodies, the following experimental controls should be incorporated:

• Positive control: Include samples known to express ZNF331 (tissue-specific, based on literature). For human samples, normal brain tissue can serve as a positive control as it shows relatively high ZNF331 expression .

• Negative control: Include samples with minimal or no ZNF331 expression or use primary antibody omission controls.

• Knockdown/knockout validation: Use siRNA/shRNA knockdown or CRISPR/Cas9 knockout cells to validate antibody specificity. The significant reduction or elimination of signal in these samples confirms specificity.

• Overexpression validation: Use cells transfected with ZNF331 expression vectors to confirm increased signal detection.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide, which should eliminate specific signals.

• Cross-reactivity testing: If the antibody claims multiple species reactivity, validate in each species independently.

• Loading controls: For western blots, include appropriate housekeeping protein controls (β-actin, GAPDH, etc.) to normalize expression levels .

What is the role of ZNF331 in the PABPC5/HCG15/ZNF331 feedback loop, and how might this impact antibody-based detection methods?

The PABPC5/HCG15/ZNF331 feedback loop represents a complex regulatory mechanism with significant implications for both cancer biology and antibody-based detection strategies:

• Mechanism of the feedback loop:

  • PABPC5 binds to HCG15 and increases its stability

  • HCG15 promotes the degradation of ZNF331 mRNA through the SMD (Staufen-mediated mRNA decay) pathway

  • ZNF331 binds to the promoter regions of both LAMC2 and PABPC5, inhibiting their transcription

  • This creates a negative feedback loop where ZNF331 suppresses PABPC5, which indirectly increases ZNF331 levels

• Impact on antibody-based detection:

  • Expression levels of ZNF331 may fluctuate based on the status of other components in this feedback loop

  • In experimental systems where PABPC5 or HCG15 levels are manipulated, researchers should anticipate corresponding changes in ZNF331 expression

  • When knockdown studies are performed (e.g., PABPC5 knockdown), ZNF331 mRNA and protein expression increase significantly

  • Conversely, HCG15 overexpression reduces ZNF331 mRNA and protein levels

• Methodological considerations:

  • When performing ZNF331 antibody-based detection, the status of PABPC5 and HCG15 should be considered as potential confounding factors

  • Analysis of all three components may be necessary for comprehensive understanding

  • Different cell types and cancer states may exhibit variations in this regulatory loop's function

How can epitope masking or protein-protein interactions affect ZNF331 antibody detection?

Several molecular phenomena can impact ZNF331 antibody detection efficiency and specificity:

• Epitope masking concerns:

  • ZNF331 contains multiple zinc finger domains and a KRAB domain that may participate in protein-protein interactions or DNA binding

  • The KRAB domain is known to interact with transcriptional co-repressors, which could mask antibody epitopes in native conditions

  • ZNF331 binding to promoter regions of target genes (such as LAMC2 and PABPC5) may alter protein conformation and epitope accessibility

• Methodological approaches to address epitope masking:

  • Use multiple antibodies targeting different epitopes of ZNF331

  • Compare native versus denaturing conditions in protein detection assays

  • For immunoprecipitation studies, consider crosslinking approaches to capture transient interactions

  • When performing ChIP assays to study ZNF331-DNA interactions, optimize fixation conditions to balance between preserving interactions and maintaining epitope accessibility

• Post-translational modifications:

  • Consider how post-translational modifications might affect antibody recognition

  • Use phospho-specific antibodies if studying signaling-dependent regulation of ZNF331 function

  • When studying nuclear localization, consider how PTMs might regulate subcellular distribution

What are the optimal immunoprecipitation conditions for studying ZNF331 protein-protein interactions or chromatin binding?

Based on research methodologies used in ZNF331 studies, the following optimized conditions for immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) are recommended:

For standard immunoprecipitation (protein-protein interactions):

• Lysis buffer: RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0) supplemented with protease inhibitors

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Use 2-5 μg of ZNF331 antibody per 500 μg of total protein

• Incubation time: Overnight at 4°C with gentle rotation

• Washing: 4-5 washes with decreasing salt concentration to maintain specific interactions while removing non-specific binding

For chromatin immunoprecipitation (DNA-protein interactions):

• Fixation: 1% formaldehyde for 10 minutes at room temperature

• Sonication conditions: Optimize to achieve chromatin fragments of 200-500 bp

• Antibody amount: 5 μg of ZNF331 antibody per ChIP reaction

• Include appropriate controls: IgG negative control and positive control for known ZNF331 binding sites

• For analysis of ZNF331 binding to LAMC2 or PABPC5 promoters, design primers spanning the predicted binding sites

These conditions have been successfully employed to demonstrate ZNF331 binding to promoter regions of target genes like LAMC2 and PABPC5, providing insights into the transcriptional repression function of ZNF331 .

What are common reasons for non-specific binding or poor signal when using ZNF331 antibodies?

When researchers encounter issues with ZNF331 antibody performance, several factors may contribute to non-specific binding or weak signal:

• Non-specific binding issues:

  • Insufficient blocking: Increase blocking time or use alternative blocking agents (5% BSA or 5% milk)

  • High antibody concentration: Titrate antibody to determine optimal dilution

  • Cross-reactivity with related zinc finger proteins: Validate antibody specificity using knockdown controls

  • Secondary antibody cross-reactivity: Use secondary antibodies specifically matched to host species

• Weak or absent signal issues:

  • Low target expression: ZNF331 is variably expressed across tissues and commonly downregulated in cancer samples

  • Epitope masking: Consider native versus denaturing conditions

  • Antibody degradation: Avoid repeated freeze-thaw cycles

  • Inappropriate application: Verify the antibody is validated for your specific application

• Optimization strategies:

  • For Western blotting: Optimize protein extraction methods, especially for nuclear proteins

  • For IHC/ICC: Test multiple antigen retrieval methods (heat-induced versus enzymatic)

  • For all applications: Compare results from multiple antibodies targeting different epitopes of ZNF331

How should researchers design experiments to study the relationship between ZNF331 expression and biological outcomes in cancer models?

To effectively investigate ZNF331's role in cancer biology, experiments should be designed with these key considerations:

• Expression analysis approach:

  • Compare paired tumor/normal samples from the same patients when possible

  • Analyze ZNF331 expression across cancer progression stages (normal → low-grade → high-grade)

  • Use multiple detection methods: qPCR for mRNA, Western blotting and IHC for protein

• Functional studies design:

  • Construct both knockdown and overexpression models:

    • For knockdown: siRNA/shRNA with validated efficiency (>70% reduction)

    • For overexpression: Use vectors with appropriate promoters (aim for 2-3× increase)

  • Include appropriate controls: scrambled siRNA, empty vectors

• Biological outcomes assessment:

  • Proliferation: Cell Counting Kit-8 or similar assays

  • Migration/invasion: Transwell assays

  • Vasculogenic mimicry: In vitro VM tube formation assays

• Mechanistic studies:

  • ChIP assays to identify direct gene targets

  • RNA-seq following ZNF331 modulation to identify broader transcriptional impacts

  • Co-immunoprecipitation to identify protein interaction partners

• Recommended experimental design structure:

  • First establish expression patterns in clinical samples

  • Validate findings in cell line models

  • Perform gain/loss of function studies

  • Investigate downstream molecular mechanisms

  • Connect to clinically relevant outcomes using in vivo models when possible

How can researchers differentiate between the effects of ZNF331 and other zinc finger proteins with similar domains?

Due to the large zinc finger protein family with similar structural domains, carefully designed experimental approaches are essential to establish ZNF331-specific functions:

• Sequence-based specificity:

  • Design siRNAs targeting unique regions of ZNF331 mRNA

  • Validate knockdown specificity by measuring expression of related zinc finger proteins

  • For antibody-based detection, select antibodies raised against unique regions outside conserved zinc finger domains

• Rescue experiments:

  • Perform knockdown of endogenous ZNF331 followed by re-expression of siRNA-resistant ZNF331 constructs

  • If the phenotype is specifically due to ZNF331 loss, re-expression should rescue the effect

  • Include expression of related zinc finger proteins as controls to demonstrate specificity

• Domain-specific analysis:

  • Create domain deletion or mutation constructs (ΔKRAB, zinc finger mutations)

  • Assess which domains are essential for observed phenotypes

  • Compare with similar domains from related proteins

• Target gene specificity:

  • Identify ZNF331-specific target genes through ChIP-seq

  • Confirm direct binding through reporter assays with wild-type and mutated binding sites

  • Demonstrate that these targets are not regulated by closely related zinc finger proteins

• Use CRISPR/Cas9 for gene editing:

  • Generate clean knockout cell lines for definitive loss-of-function studies

  • Create knock-in cell lines with tagged ZNF331 for more specific detection

  • Use precise editing to modify specific domains while leaving others intact

What are the most effective strategies for multiplexed detection of ZNF331 alongside other components of the PABPC5/HCG15/ZNF331 regulatory loop?

Investigating the complex PABPC5/HCG15/ZNF331 regulatory loop requires sophisticated multiplexed detection approaches:

• Multi-color immunofluorescence:

  • Use primary antibodies from different host species (e.g., rabbit anti-ZNF331, mouse anti-PABPC5)

  • Select fluorophore-conjugated secondary antibodies with minimal spectral overlap

  • Include DAPI for nuclear counterstaining

  • Analyze colocalization using appropriate software (ImageJ with colocalization plugins)

• Sequential immunoprecipitation approaches:

  • First IP: Pull down one component (e.g., ZNF331)

  • Second IP: Use eluate from first IP to pull down interaction partners

  • Western blot analysis of final eluate for all components

  • Controls should include single IPs and IgG controls

• RNA-protein interaction analysis:

  • RNA immunoprecipitation (RIP) to study HCG15 binding to PABPC5

  • Use crosslinking immunoprecipitation (CLIP) for enhanced specificity

  • qRT-PCR analysis of precipitated RNA

  • Include controls for non-specific RNA binding

• Chromatin interaction studies:

  • ChIP-reChIP to identify genomic regions bound by multiple factors

  • Sequential ChIP first with ZNF331 antibody followed by PABPC5 antibody

  • qPCR or sequencing of precipitated DNA focusing on promoter regions of interest

• Live-cell imaging approaches:

  • Create fluorescent protein fusions (ensuring functionality is preserved)

  • Perform fluorescence recovery after photobleaching (FRAP) to study dynamics

  • Use proximity ligation assay (PLA) to visualize protein-protein interactions in situ

How can ZNF331 antibodies be utilized in the development of prognostic or diagnostic biomarkers for glioma or other cancers?

The potential of ZNF331 as a biomarker can be explored through these methodological approaches:

• Tissue microarray analysis:

  • Develop standardized IHC protocols with ZNF331 antibodies

  • Use digital pathology for quantitative assessment

  • Establish scoring criteria (percentage positive cells, staining intensity)

  • Correlate with clinical outcomes (survival, treatment response)

• Biomarker validation process:

  • Discovery phase: Compare ZNF331 expression across different cancer types and stages

  • Validation phase: Confirm findings in independent patient cohorts

  • Clinical utility testing: Determine if ZNF331 status affects clinical decision-making

• Multimarker panel development:

  • Combine ZNF331 with other components of the regulatory loop (PABPC5, HCG15)

  • Include established biomarkers for the specific cancer type

  • Use statistical methods to determine the optimal marker combination

  • Calculate sensitivity, specificity, and AUC for the panel

• Sample considerations:

  • Evaluate ZNF331 in different sample types (FFPE tissue, fresh frozen, liquid biopsies)

  • Assess pre-analytical variables affecting detection (fixation time, processing methods)

  • Determine minimal sample requirements for reliable detection

• From research to clinical application:

  • Standardize antibody-based detection methods across laboratories

  • Develop reference materials for quality control

  • Design prospective clinical trials to validate clinical utility

  • Consider companion diagnostic development if ZNF331 status predicts therapy response

What methodological approaches should be used when investigating the epigenetic regulation of ZNF331 expression in different cellular contexts?

Given evidence of ZNF331 promoter methylation in gastric cancer, comprehensive epigenetic analysis requires these methodological considerations:

• DNA methylation analysis:

  • Bisulfite sequencing of the ZNF331 promoter region:

    • Design primers that exclude CpG sites to prevent bias

    • Include both proximal promoter and distal regulatory elements

    • Analyze at least 10 clones per sample for accurate methylation patterns

  • Methylation-specific PCR (MSP) for targeted analysis of key CpG sites

  • Quantitative approaches: Pyrosequencing or methylation arrays for high-throughput analysis

• Histone modification analysis:

  • ChIP assays using antibodies against specific histone marks:

    • Repressive marks: H3K27me3, H3K9me3

    • Active marks: H3K4me3, H3K27ac

  • Sequential ChIP to determine co-occurrence of modifications

  • ChIP-seq for genome-wide analysis of histone modifications at the ZNF331 locus

• Functional analysis of epigenetic regulation:

  • Treatment with epigenetic modifying drugs:

    • DNA methyltransferase inhibitors (5-aza-2'-deoxycytidine)

    • Histone deacetylase inhibitors (TSA, SAHA)

  • Monitor ZNF331 expression changes using qRT-PCR and Western blotting

  • Use chromatin accessibility assays (ATAC-seq, DNase-seq) to assess regulatory regions

• Circular chromosome conformation capture (4C) or Hi-C:

  • Identify long-range chromatin interactions affecting ZNF331 regulation

  • Focus on interactions between the ZNF331 promoter and potential enhancers

• Single-cell approaches:

  • Single-cell RNA-seq coupled with single-cell ATAC-seq

  • Correlate ZNF331 expression with chromatin accessibility at single-cell resolution

  • Identify cell-type specific regulatory mechanisms

What considerations should be made when developing ZNF331 antibodies for therapeutic applications targeting the PABPC5/HCG15/ZNF331 pathway in glioma?

While primarily used as research tools, antibodies against components of the PABPC5/HCG15/ZNF331 pathway may have therapeutic potential. Key considerations include:

• Target selection and validation:

  • Determine whether to target ZNF331 directly or other pathway components

  • For glioma, increasing ZNF331 function appears beneficial based on its tumor suppressor activity

  • Validate therapeutic hypothesis using genetic approaches before antibody development

  • Consider targeting upstream regulators (PABPC5, HCG15) that suppress ZNF331

• Antibody format considerations:

  • Blood-brain barrier (BBB) penetration is critical for glioma applications:

    • Consider smaller formats (Fab, scFv, nanobodies) for improved BBB penetration

    • Evaluate BBB-crossing technologies (receptor-mediated transcytosis)

  • For intracellular targets like ZNF331, develop cell-penetrating antibodies or antibody-drug conjugates

  • Consider bispecific formats targeting both the tumor cells and immune effector cells

• Delivery challenges and strategies:

  • Direct administration methods for brain tumors:

    • Convection-enhanced delivery

    • Intratumoral injection

    • Use of implantable devices for sustained release

  • Nanoparticle formulations for improved delivery

  • Combination with BBB disruption techniques (focused ultrasound, osmotic disruption)

• Functional testing hierarchy:

  • In vitro: Cell line models with defined ZNF331 status

  • Ex vivo: Patient-derived organoids or explants

  • In vivo: Orthotopic glioma models with appropriate microenvironment

• Safety and efficacy considerations:

  • Off-target effects on normal cells expressing ZNF331

  • Effects on other zinc finger proteins with structural similarity

  • Impact on global transcriptional networks

  • Potential immunogenicity of novel antibody formats

What protocol modifications are needed when using ZNF331 antibodies for chromatin immunoprecipitation sequencing (ChIP-seq) experiments?

ChIP-seq for ZNF331 requires specific optimizations to account for the protein's transcription factor properties:

• Crosslinking optimization:

  • Standard protocol: 1% formaldehyde for 10 minutes at room temperature

  • For ZNF331: Consider dual crosslinking approach:

    • First crosslink with protein-protein crosslinker (e.g., disuccinimidyl glutarate, DSG)

    • Follow with formaldehyde crosslinking

  • This approach better preserves interactions between ZNF331 and cofactors or histones

• Chromatin fragmentation considerations:

  • Target fragment size: 200-500 bp

  • For ZNF331, which binds to specific DNA motifs:

    • Use enzymatic digestion (e.g., MNase) for more precise fragmentation around binding sites

    • If using sonication, optimize conditions to minimize epitope destruction

• Antibody selection and validation:

  • Pre-screen multiple ZNF331 antibodies using ChIP-qPCR at known target sites (LAMC2, PABPC5 promoters)

  • Validate antibody specificity using ZNF331 knockout/knockdown controls

  • For ChIP-seq, use antibodies that recognize native (non-denatured) ZNF331

• IP protocol modifications:

  • Increase antibody amount: 5-10 μg per ChIP reaction

  • Extended incubation: Overnight at 4°C with rotation

  • Include stringent washes to reduce background

  • Consider sequential ChIP for studying ZNF331 co-localization with other factors

• Library preparation and sequencing:

  • Input normalization is critical

  • Include spike-in controls for normalization across samples

  • Higher sequencing depth (>30 million reads) may be required for precise motif identification

  • Paired-end sequencing recommended for better peak resolution

How should researchers design experiments to identify and validate direct transcriptional targets of ZNF331?

A comprehensive approach to identifying ZNF331 transcriptional targets should combine multiple complementary methods:

• Genome-wide binding site identification:

  • ChIP-seq to map all ZNF331 binding sites across the genome

  • ATAC-seq or DNase-seq to correlate binding with chromatin accessibility

  • Motif analysis to identify ZNF331 binding consensus sequences

  • For validation, use electrophoretic mobility shift assays (EMSA) with recombinant ZNF331

• Gene expression profiling after ZNF331 modulation:

  • RNA-seq following ZNF331 knockdown and overexpression

  • Time-course experiments to distinguish direct vs. indirect targets

  • Consider using inducible expression systems for temporal control

  • Integrate with ChIP-seq data to identify direct targets (genes with both binding and expression changes)

• Functional validation of individual targets:

  • Luciferase reporter assays with wild-type and mutated ZNF331 binding sites

  • Design workflow:

    • Clone promoter regions with ZNF331 binding sites into reporter vectors

    • Test activity with/without ZNF331 expression

    • Mutate binding sites to confirm specificity

  • CRISPR interference or activation at specific binding sites

• Mechanistic studies of transcriptional regulation:

  • Assess repression vs. activation functions through reporter assays

  • Investigate recruitment of co-repressors (especially for KRAB domain function)

  • Study histone modifications at target promoters after ZNF331 binding

  • Analyze DNA methylation changes at binding sites

• Integration with existing databases:

  • Compare identified targets with known ZNF331-regulated genes

  • Pathway enrichment analysis of target genes

  • Cross-reference with cancer-related gene sets

What strategies can researchers use to study the post-translational modifications of ZNF331 and their impact on protein function?

Post-translational modifications (PTMs) can significantly affect ZNF331 function. A systematic approach to studying these modifications includes:

• Identification of PTM sites:

  • Mass spectrometry-based approaches:

    • Immunoprecipitate ZNF331 under native conditions

    • Analyze using LC-MS/MS with PTM-specific methods

    • Use both bottom-up (peptide) and top-down (intact protein) approaches

  • Targeted analysis for specific modifications:

    • Phosphorylation: Phospho-enrichment followed by MS

    • Ubiquitination: Use tandem ubiquitin binding entities (TUBEs)

    • SUMOylation: Use SUMO-specific enrichment techniques

• Functional analysis of identified PTMs:

  • Site-directed mutagenesis of modified residues:

    • Phospho-mimetic mutations (S/T to E/D)

    • Phospho-null mutations (S/T to A)

    • Lysine mutations for ubiquitination/SUMOylation sites (K to R)

  • Compare wild-type and mutant ZNF331 for:

    • DNA binding activity (ChIP, EMSA)

    • Transcriptional repression function (reporter assays)

    • Protein stability and half-life

    • Subcellular localization

• PTM-specific antibody development and validation:

  • Generate phospho-specific antibodies for key modified sites

  • Validate using phosphatase treatment controls

  • Apply in western blotting, immunofluorescence to track modification status

• Analysis of PTM regulation:

  • Identify kinases/phosphatases for phosphorylation sites

  • Study E3 ligases/deubiquitinases for ubiquitination

  • Investigate conditions affecting modification status:

    • Cell cycle phases

    • Stress responses

    • Cancer-related signaling pathways

• Structural impact analysis:

  • In silico modeling of how PTMs affect protein structure

  • Assess impact on zinc finger domain conformation

  • Study effects on protein-protein interactions