ZNF77 Antibody

What is ZNF77 and what is its biological function?

ZNF77 belongs to the krueppel C2H2-type zinc-finger protein family and functions as a transcription factor. It contains 12 C2H2-type zinc fingers and 1 KRAB domain, suggesting a role in transcriptional regulation . Bioinformatics modeling indicates that ZNF77 may control the expression of defensins, elastase, and calmodulin, which are potentially important for fungal clearance in the lung epithelium . Recent research has identified ZNF77 as a key controller of Aspergillus colonization in the respiratory airways, making it potentially valuable as a risk-marker for patient stratification .

What types of ZNF77 antibodies are currently available for research?

Current research-grade ZNF77 antibodies are primarily rabbit polyclonal antibodies. These antibodies are typically generated against specific immunogen sequences, such as "RPCKECGQACSCLSCQSPPMKTQTVEKPCNCQDSRTASVTYVKSLSSKKSYECQKCGKAFICPSS," which corresponds to portions of the human ZNF77 protein . These antibodies are designed for applications including immunohistochemistry (IHC), immunocytochemistry/immunofluorescence (ICC/IF), and Western blotting (WB) .

What are the standard applications for ZNF77 antibodies in research?

ZNF77 antibodies are validated for several standard research applications:

These applications enable researchers to study ZNF77 expression patterns, subcellular localization, and protein levels in various experimental contexts .

What is the relationship between ZNF77 and respiratory diseases?

Recent studies have established that ZNF77, particularly the genetic variant rs35699176, influences the integrity of the bronchial epithelium. This variant causes a loss of epithelial integrity and increases levels of extracellular matrix proteins, promoting Aspergillus fumigatus conidial adhesion, germination, and growth . Patients with fungal asthma who carry the rs35699176 variant show higher A. fumigatus loads in their respiratory airways, suggesting ZNF77's importance in controlling fungal colonization in conditions like Allergic Bronchopulmonary Aspergillosis (ABPA) .

How should researchers optimize immunohistochemistry protocols for ZNF77 detection in lung tissue samples?

For optimal ZNF77 detection in lung tissue samples, researchers should consider the following protocol modifications:

• Fixation: Use 10% neutral buffered formalin for 24-48 hours, as overfixation can mask epitopes

• Antigen retrieval: Test both heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) and EDTA buffer (pH 9.0) to determine optimal conditions

• Blocking: Employ a dual blocking approach with both serum-free protein block and avidin/biotin blocking kit if using biotinylated secondary antibodies

• Primary antibody: Incubate with anti-ZNF77 antibody at dilutions between 1:50-1:200 overnight at 4°C

• Detection system: Compare DAB and AEC chromogens to determine optimal visualization, especially when studying lung tissues with potential endogenous pigmentation

• Counterstaining: Use light hematoxylin counterstaining to avoid obscuring nuclear ZNF77 signals

Validation should include both positive controls (tissues known to express ZNF77) and negative controls (primary antibody omission and isotype controls).

What are the technical challenges in detecting ZNF77 protein interactions in co-immunoprecipitation experiments?

Several technical challenges exist when studying ZNF77 protein interactions:

• Transcription factor solubility: As a transcription factor, ZNF77 may be tightly bound to chromatin. Researchers should optimize nuclear extraction protocols using different salt concentrations (0.3-0.5M NaCl) and detergent combinations to maximize solubilization without disrupting protein-protein interactions .

• Cross-linking considerations: Due to potentially transient interactions, consider using reversible cross-linking agents like DSP (dithiobis(succinimidyl propionate)) before immunoprecipitation.

• Antibody orientation: Test both N-terminal and C-terminal targeting antibodies, as the zinc finger domains might interfere with antibody access in protein complexes.

• Validation approaches: Employ reciprocal co-IP (pulling down with the interacting protein antibody) and proximity ligation assays as complementary methods to confirm interactions.

• Negative controls: Include IgG controls matched to the host species of the ZNF77 antibody and, when possible, ZNF77-knockout or knockdown samples.

How can researchers distinguish between specific and non-specific binding when using ZNF77 antibodies in Western blotting?

To ensure specificity in Western blotting applications with ZNF77 antibodies:

• Expected molecular weight: ZNF77 has a predicted molecular weight that should be confirmed in Western blots. Bands at unexpected molecular weights may represent non-specific binding, post-translational modifications, or isoforms .

• Validation controls:

  • Positive control: Use cell lines known to express ZNF77 (e.g., certain epithelial cell lines)

  • Negative control: Include samples with ZNF77 knockdown/knockout

  • Blocking peptide: Pre-incubate antibody with the immunogen peptide to confirm specificity

• Optimization strategies:

  • Test multiple antibody dilutions to find optimal signal-to-noise ratio

  • Optimize blocking conditions (5% milk vs. BSA) to reduce background

  • Increase washing stringency by adding 0.1-0.3% Tween-20 to wash buffers

  • For nuclear proteins like ZNF77, ensure complete protein denaturation with SDS and reducing agents

• Cross-reactivity assessment: When studying across species, account for sequence homology differences (e.g., mouse - 39%, rat - 41% identity to human) by validating with species-specific positive controls .

What methodology should researchers employ to study ZNF77's transcriptional regulatory function?

To investigate ZNF77's function as a transcription factor:

• DNA binding analysis:

  • Chromatin Immunoprecipitation (ChIP) using validated ZNF77 antibodies followed by sequencing (ChIP-seq) or qPCR of candidate target genes

  • Electrophoretic Mobility Shift Assay (EMSA) with recombinant ZNF77 or nuclear extracts and antibody supershifts

  • DNA pull-down assays with biotinylated candidate binding sequences

• Transcriptional activity assessment:

  • Luciferase reporter assays with promoters of putative target genes (e.g., defensins, elastase, and calmodulin)

  • Gene expression analysis after ZNF77 overexpression or knockdown/knockout

  • RNA-seq analysis coupled with ChIP-seq to correlate binding with expression changes

• Protein-protein interaction studies:

  • Co-IP to identify interaction with transcriptional co-factors

  • Mammalian two-hybrid assays to confirm direct interactions

  • Mass spectrometry of immunoprecipitated complexes to identify novel interaction partners

• Functional domain analysis:

  • Generate truncation or point mutations in the zinc finger domains and KRAB domain

  • Assess the impact on DNA binding, protein interactions, and transcriptional regulation

How can ZNF77 antibodies be used to study the rs35699176 variant's effects on bronchial epithelium integrity?

The rs35699176 variant introduces a premature stop codon in ZNF77 before the DNA binding region, potentially affecting its function . Researchers can employ ZNF77 antibodies to:

• Comparative expression analysis:

  • Immunohistochemistry or immunofluorescence staining of bronchial biopsy specimens from patients with and without the rs35699176 variant

  • Western blot analysis to quantify protein expression levels and detect truncated protein forms

• Functional epithelial barrier studies:

  • Immunostaining for ZNF77 alongside tight junction proteins (claudins, occludin, ZO-1) in airway epithelial models

  • Correlation of ZNF77 localization with transepithelial electrical resistance (TEER) measurements

• Genome editing approaches:

  • CRISPR/Cas9-edited bronchial epithelial cell lines with rs35699176 variant introduction

  • Antibody-based validation of edited cells by Western blot and immunofluorescence

  • Rescue experiments with wild-type ZNF77 expression

• Extracellular matrix assessment:

  • Co-staining for ZNF77 and ECM proteins (e.g., collagens, fibronectin) that are upregulated in cells with the rs35699176 variant

  • Analysis of secretome changes using antibody arrays or mass spectrometry

What are the optimal methods for using ZNF77 antibodies to investigate its role in controlling Aspergillus fumigatus colonization?

To investigate ZNF77's role in A. fumigatus colonization:

• Infection model analysis:

  • Immunostaining of in vitro bronchial epithelial infection models with A. fumigatus

  • Correlation of ZNF77 expression/localization with fungal adhesion, germination, and hyphal extension

  • Time-course analysis of ZNF77 expression during infection progression

• Patient sample studies:

  • Immunohistochemistry of bronchial biopsies from patients with and without fungal colonization

  • Correlation of ZNF77 expression patterns with fungal burden quantification

  • Stratification by rs35699176 genotype to assess genotype-phenotype relationships

• Mechanism investigation:

  • ChIP-seq analysis to identify ZNF77 binding sites in genes related to epithelial barrier function and antifungal defense

  • RNA-seq of ZNF77 wild-type vs. mutant cells to identify differentially expressed genes during A. fumigatus challenge

  • Proteomics analysis of secreted factors influenced by ZNF77 expression that impact fungal growth

• Therapeutic exploration:

  • Screening compounds that modulate ZNF77 activity using antibody-based readouts

  • Assessment of interventions that might correct rs35699176-associated defects

How can ZNF77 antibodies be integrated into biomarker development for respiratory diseases?

For biomarker development applications:

• Tissue microarray analysis:

  • Immunohistochemical staining of respiratory disease tissue microarrays with ZNF77 antibodies

  • Correlation with disease severity, progression, and treatment response

  • Development of standardized scoring systems for ZNF77 expression patterns

• Liquid biopsy approaches:

  • Detection of ZNF77 protein in bronchoalveolar lavage (BAL) fluid using customized immunoassays

  • Correlation of ZNF77 levels with fungal burden in respiratory samples

  • Multiplex analysis with other biomarkers of epithelial integrity and inflammation

• Companion diagnostic development:

  • Standardization of immunohistochemical protocols for potential clinical application

  • Comparison of ZNF77 protein expression with rs35699176 genotyping results

  • Assessment of ZNF77 as a predictive marker for antifungal treatment response

• Multimodal biomarker strategy:

  • Combination of ZNF77 antibody-based detection with genetic testing for rs35699176

  • Integration with other markers of epithelial dysfunction and fungal colonization

  • Development of risk scoring systems for patients with respiratory diseases

What are common sources of variability in ZNF77 antibody experiments and how can they be addressed?

Researchers commonly encounter several sources of variability when working with ZNF77 antibodies:

• Antibody lot-to-lot variation:

  • Run validation tests with each new lot

  • Maintain a reference sample set for standardization

  • Consider pooling antibody lots for long-term studies

• Sample preparation inconsistencies:

  • Standardize fixation times and conditions for IHC/IF

  • Optimize cell lysis buffers for complete extraction of nuclear proteins

  • Use fresh samples whenever possible, as ZNF77 may degrade during storage

• Technical variations:

  • Employ automated staining platforms for IHC/IF when available

  • Use internal loading controls for Western blots

  • Include technical replicates in all experiments

• Biological variation:

  • Account for cell cycle effects, as transcription factor expression may vary

  • Consider tissue heterogeneity when analyzing respiratory samples

  • Control for disease state and medication effects in patient samples

How should researchers approach contradictory results between different ZNF77 antibody-based detection methods?

When facing contradictory results:

• Systematic validation approach:

  • Verify antibody specificity using knockout/knockdown controls in each system

  • Test multiple antibodies targeting different epitopes of ZNF77

  • Confirm findings with non-antibody-based methods (e.g., mRNA expression)

• Method-specific considerations:

  • For Western blot vs. IHC discrepancies: Consider protein denaturation effects on epitope accessibility

  • For IHC vs. IF differences: Evaluate fixation effects and detection sensitivity

  • For in vitro vs. in vivo disparities: Account for microenvironmental factors

• Data integration strategies:

  • Weight evidence based on strength of controls and technical rigor

  • Consider biological context when interpreting conflicting results

  • Develop hypotheses that might explain genuine biological differences detected by different methods

• Reporting recommendations:

  • Transparently document all methodological details

  • Report both confirming and contradictory findings

  • Discuss limitations of each detection method

What are the best practices for antibody validation when studying ZNF77 in novel experimental systems?

When introducing ZNF77 antibodies to new experimental systems:

• Specificity validation:

  • CRISPR/Cas9 knockout or siRNA knockdown controls

  • Overexpression of tagged ZNF77 for co-localization studies

  • Immunoprecipitation followed by mass spectrometry identification

• Cross-reactivity assessment:

  • Western blot analysis for off-target bands

  • Peptide competition assays

  • Testing in samples known to lack ZNF77 expression

• Application-specific validation:

  • For IHC: Optimize antigen retrieval and test multiple antibody concentrations

  • For IF: Compare fixation methods and test permeabilization conditions

  • For IP-based applications: Optimize lysis conditions for complete extraction

• System-specific considerations:

  • For primary cells: Compare with established cell lines

  • For tissue explants: Account for potential autofluorescence

  • For animal models: Consider species homology (human-mouse: 39%, human-rat: 41%)

How might ZNF77 antibodies contribute to understanding the broader role of zinc finger proteins in respiratory health?

ZNF77 research may provide insights into the wider family of zinc finger proteins:

• Comparative analysis approaches:

  • Parallel staining with antibodies against multiple ZNF family members in respiratory tissues

  • Characterization of differential expression patterns in health vs. disease

  • Investigation of potential cooperative or competitive functions

• Evolutionary significance:

  • Cross-species comparison of ZNF77 expression and function

  • Investigation of selection pressure on ZNF77 in populations with different respiratory pathogen exposures

  • Correlation of genetic variants with protein expression and function

• Transcriptional network mapping:

  • Integration of ChIP-seq data from multiple ZNF family members

  • Construction of transcriptional regulatory networks in respiratory epithelium

  • Identification of shared and unique target genes among related ZNF proteins

• Therapeutic implications:

  • Target validation for small molecule development

  • Assessment of ZNF77 modulation as a potential approach to strengthen epithelial barrier function

  • Development of engineered ZNF proteins with enhanced antifungal properties

What novel approaches might enhance the sensitivity and specificity of ZNF77 detection in clinical samples?

Emerging technologies for improved ZNF77 detection include:

• Advanced antibody engineering:

  • Single-domain antibodies (nanobodies) for improved tissue penetration

  • Recombinant antibody fragments with enhanced specificity

  • Bispecific antibodies targeting ZNF77 alongside interacting partners

• Proximity-based detection methods:

  • Proximity ligation assays (PLA) to detect ZNF77 interactions with DNA or proteins

  • Proximity extension assays for ultrasensitive detection in limited samples

  • CODEX multiplexed imaging for single-cell spatial analysis of ZNF77 and multiple markers

• Digital pathology integration:

  • Machine learning algorithms for automated quantification of ZNF77 staining patterns

  • Whole slide imaging analysis for heterogeneity assessment

  • Integration of genotype data with protein expression patterns

• Single-cell approaches:

  • Single-cell Western blot for ZNF77 quantification

  • Mass cytometry (CyTOF) with metal-conjugated ZNF77 antibodies

  • Spatial transcriptomics combined with protein detection

How can multi-omics approaches incorporating ZNF77 antibody data advance personalized medicine for respiratory diseases?

Integration of ZNF77 antibody data with other -omics approaches:

• Integrative analysis frameworks:

  • Correlation of ZNF77 protein levels with genotype, transcriptome, and proteome data

  • Patient stratification based on integrated molecular profiles

  • Development of predictive models for fungal colonization risk

• Longitudinal monitoring strategies:

  • Serial sampling of respiratory specimens with ZNF77 quantification

  • Tracking changes in ZNF77 expression during disease progression or treatment

  • Correlation with clinical outcomes and treatment response

• Personalized intervention approaches:

  • Tailoring antifungal prophylaxis based on ZNF77 status

  • Development of targeted therapies for patients with rs35699176 variant

  • Biomarker-guided clinical trial design

• Implementation science considerations:

  • Cost-effectiveness analysis of ZNF77 testing

  • Development of standardized clinical assays

  • Integration into clinical decision support systems