ZNF829 Antibody

What is ZNF829 and why is it important in research?

ZNF829 (Zinc Finger Protein 829) is a nuclear protein encoded by a gene located on chromosome 19q13.12. It functions as a DNA-binding transcription factor with RNA polymerase II-specific activity and is predicted to be involved in the regulation of transcription by RNA polymerase II . ZNF829 contains zinc finger domains, which are critical for its DNA-binding capabilities. The protein's role in transcriptional regulation makes it a subject of interest in studies investigating gene expression control mechanisms, particularly in human cells. Research on ZNF829 contributes to our understanding of fundamental transcriptional regulatory networks and potentially disease-related gene expression patterns.

What are the primary applications of ZNF829 antibodies in research?

ZNF829 antibodies are primarily utilized in techniques that detect, quantify, or isolate the ZNF829 protein in experimental systems. The most common applications include:

• Western Blotting (WB): For detection and semi-quantitative analysis of ZNF829 protein expression in cell or tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of ZNF829 protein levels

• Flow Cytometry (FACS): For detection of ZNF829 in individual cells, particularly useful for examining protein expression in heterogeneous cell populations

• Immunoprecipitation: For isolation of ZNF829 protein complexes to study protein-protein interactions

• Immunohistochemistry/Immunofluorescence: For visualization of ZNF829 localization within cells or tissues

The selection of appropriate antibody depends on the specific application, with considerations for binding region specificity, host species, and conjugation requirements.

What are the different types of ZNF829 antibodies available and how do they differ?

ZNF829 antibodies vary based on several key characteristics:

The binding region specificity is particularly important as different epitopes may be accessible in various experimental conditions or may correlate with different functional domains of the protein. Most commercially available ZNF829 antibodies are developed in rabbits and are polyclonal in nature, offering broad epitope recognition but potentially higher batch-to-batch variability compared to monoclonal antibodies .

How should I select the appropriate ZNF829 antibody for my specific research application?

Selection of the optimal ZNF829 antibody should follow a methodical evaluation process:

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, ELISA, FACS, etc.). For example, antibodies targeting AA 127-155 have been validated for Western Blotting and Flow Cytometry, while those targeting AA 133-432 are often recommended for ELISA applications .

• Epitope consideration: Select antibodies targeting epitopes relevant to your research question. If studying a specific domain of ZNF829, choose antibodies binding to that region. The central region (AA 127-155) may be more accessible in native protein configurations, while antibodies targeting AA 133-432 recognize a larger portion of the protein .

• Conjugation requirements: Select unconjugated antibodies for applications requiring primary-secondary detection systems. Choose directly conjugated antibodies (HRP, FITC, Biotin) for applications benefiting from direct detection or when minimizing cross-reactivity is essential .

• Validation evidence: Review available validation data, including Western blot images, ELISA curves, or flow cytometry profiles that demonstrate specificity and sensitivity.

• Cross-reactivity profile: Confirm species reactivity matches your experimental model. Most ZNF829 antibodies react with human samples, with limited cross-reactivity to other species .

Testing multiple antibodies targeting different epitopes in parallel can provide complementary data and increase confidence in results, particularly for novel experimental systems.

What are the recommended protocols for using ZNF829 antibodies in Western blotting?

For optimal Western blotting results with ZNF829 antibodies, follow these methodological recommendations:

Sample Preparation:

• Extract total protein from cells/tissues using a lysis buffer containing protease inhibitors

• Quantify protein concentration using BCA or Bradford assay

• Denature samples at 95°C for 5 minutes in reducing Laemmli buffer

Electrophoresis and Transfer:

• Load 20-50 μg of protein per lane on a 10% SDS-PAGE gel

• Separate proteins at 100-120V until proper resolution

• Transfer to PVDF or nitrocellulose membrane (PVDF recommended for higher protein binding capacity)

Antibody Incubation:

• Block membrane in 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary ZNF829 antibody (e.g., ABIN655374, dilution 1:500-1:1000) overnight at 4°C

• Wash 3-5 times with TBST

• Incubate with appropriate HRP-conjugated secondary antibody (or use directly HRP-conjugated primary like ABIN7176554) for 1-2 hours at room temperature

• Wash 3-5 times with TBST

Detection:

• Apply chemiluminescent substrate and develop according to manufacturer's instructions

• Expected molecular weight for ZNF829 is approximately 67-72 kDa

Validation Controls:

• Include positive control (e.g., cell line known to express ZNF829)

• Consider using recombinant ZNF829 protein (e.g., NBP257673PEP) as a reference standard

• Perform peptide competition assay using the immunizing peptide to confirm specificity

Working dilutions should be empirically determined, but starting dilutions of 1:500-1:1000 for primary antibody and 1:2000-1:5000 for secondary antibody are typically recommended .

How can I optimize ZNF829 antibody-based ELISA for quantitative analysis?

For developing a robust ELISA protocol using ZNF829 antibodies:

Plate Preparation:

• Coat high-binding ELISA plates with capture antibody (e.g., unconjugated anti-ZNF829) at 1-5 μg/mL in carbonate-bicarbonate buffer (pH 9.6)

• Incubate overnight at 4°C

• Wash 3 times with PBS-T (PBS + 0.05% Tween-20)

• Block with 1-3% BSA in PBS for 1-2 hours at room temperature

Sample Processing:

• Prepare standards using recombinant ZNF829 protein (e.g., NBP257673PEP)

• Create a standard curve (typically 0-1000 ng/mL) with 2-fold serial dilutions

• Dilute samples appropriately in blocking buffer

• Add standards and samples to wells and incubate for 2 hours at room temperature

Detection:

• Wash 5 times with PBS-T

• Add biotinylated or HRP-conjugated detection antibody (e.g., ABIN7176554 or biotin-conjugated antibodies)

• For biotinylated antibodies, add streptavidin-HRP after washing

• Develop with TMB substrate and stop with 2N H₂SO₄

• Read absorbance at 450 nm

Optimization Strategies:

• Antibody pair optimization: Test different capture and detection antibody combinations targeting non-overlapping epitopes

• Titrate antibody concentrations to determine optimal signal-to-noise ratio

• Adjust sample dilutions to ensure measurements fall within the linear range of the standard curve

• Include spike-and-recovery experiments to validate detection in complex matrices

For assay validation, determine the lower limit of detection (LLOD), upper limit of quantification (ULOQ), intra- and inter-assay coefficients of variation (CV), and perform parallelism tests between recombinant standards and endogenous protein.

How can ZNF829 antibodies be effectively used in chromatin immunoprecipitation (ChIP) experiments?

While standard ChIP protocols can be adapted for ZNF829, several considerations are crucial for successful implementation:

Protocol Modifications for ZNF829 ChIP:

• Crosslinking optimization: Since ZNF829 is a transcription factor with DNA-binding activity, standard 1% formaldehyde for 10 minutes may be sufficient, but optimization (0.5-2% formaldehyde, 5-15 minutes) might be necessary.

• Antibody selection: Choose antibodies targeting regions outside the DNA-binding zinc finger domains to avoid epitope masking. Antibodies recognizing AA 127-155 or AA 133-432 may be suitable candidates .

• Chromatin fragmentation: Aim for fragments of 200-500 bp using sonication parameters optimized for your sonicator model.

• Antibody amount: Start with 3-5 μg of antibody per ChIP reaction with 25-30 μg of chromatin.

• Controls: Include:

  • Input control (non-immunoprecipitated chromatin)

  • IgG control from the same species as the ZNF829 antibody

  • Positive control (antibody against histone modification like H3K4me3)

  • Negative control (primers for a genomic region not expected to be bound by ZNF829)

Data Analysis Approach:

• Normalize ChIP-qPCR data to input and IgG controls

• For ChIP-seq, use appropriate peak calling algorithms (e.g., MACS2)

• Perform motif enrichment analysis on peaks to identify ZNF829 binding motifs

• Integrate with transcriptomic data to correlate binding with gene expression

When troubleshooting failed ChIP experiments, systematically evaluate crosslinking efficiency, antibody specificity, chromatin fragmentation quality, and washing stringency.

What are the approaches for validating ZNF829 antibody specificity in experimental systems?

Comprehensive validation of ZNF829 antibodies is critical for ensuring reliable research outcomes:

Validation Strategy 1: Genetic Approaches

• CRISPR/Cas9 knockout: Generate ZNF829 knockout cell lines and confirm absence of signal in antibody-based assays

• siRNA/shRNA knockdown: Demonstrate reduced signal intensity proportional to knockdown efficiency

• Overexpression: Show increased signal in cells overexpressing ZNF829

Validation Strategy 2: Biochemical Approaches

• Peptide competition assay: Pre-incubate antibody with immunizing peptide (e.g., recombinant ZNF829 protein NBP257673PEP) and demonstrate signal blocking

• Mass spectrometry: Confirm identity of immunoprecipitated proteins

• Parallel antibody testing: Compare results from multiple antibodies targeting different epitopes (e.g., AA 127-155 vs AA 133-432)

When encountering inconsistent results between different techniques using ZNF829 antibodies:

Analytical Framework for Resolving Discrepancies:

• Epitope accessibility analysis:

  • In Western blotting, denatured conditions expose all epitopes

  • In IP or FACS, native conformations may mask certain epitopes

  • Solution: Test antibodies targeting different regions (e.g., AA 127-155, AA 133-432, AA 87-136)

• Post-translational modification interference:

  • Phosphorylation, ubiquitination, or other PTMs may affect antibody binding

  • Solution: Use phosphatase treatment before Western blotting or IP to determine if PTMs impact detection

• Isoform-specific detection:

  • ZNF829 may have multiple isoforms differentially recognized by antibodies

  • Solution: Analyze protein databases for isoform information and select antibodies detecting all or specific isoforms

• Cross-reactivity assessment:

  • Non-specific binding may create false positives in less stringent applications

  • Solution: Increase washing stringency and use multiple antibodies targeting different epitopes

• Sensitivity threshold differences:

  • Western blotting may be more/less sensitive than ELISA or FACS

  • Solution: Optimize protein loading/concentration and detection methods for each technique

Case Study Approach to Methodological Reconciliation:
When Western blot shows a clear band but immunofluorescence shows no signal:

• Optimize fixation methods (try both paraformaldehyde and methanol fixation)

• Test antigen retrieval methods to expose potentially masked epitopes

• Use different antibody concentrations (typically higher for IF than WB)

• Consider subcellular localization (nuclear proteins may require special permeabilization)

Creating a comprehensive troubleshooting decision tree specific to your experimental system can systematically address discrepancies.

How is ZNF829 being studied in the context of transcriptional regulation networks?

ZNF829's role as a predicted DNA-binding transcription factor positions it as an important component in transcriptional regulatory networks . Current research approaches include:

Integrative Genomic Approaches:

• ChIP-seq combined with RNA-seq: Correlating ZNF829 binding sites with gene expression changes

• HiChIP or ChIA-PET: Investigating three-dimensional chromatin interactions mediated by ZNF829

• ATAC-seq with ZNF829 perturbation: Examining changes in chromatin accessibility following ZNF829 modulation

Protein Interaction Studies:

• Proximity labeling (BioID/APEX): Identifying proteins in close proximity to ZNF829

• Co-immunoprecipitation with mass spectrometry: Characterizing ZNF829 protein complexes

• Yeast two-hybrid screening: Identifying direct protein interaction partners

Functional Genomic Screens:

• CRISPR activation/interference at ZNF829 binding sites: Determining functional relevance of binding

• shRNA libraries targeting ZNF829 cofactors: Identifying cooperative transcriptional regulators

• Single-cell RNA-seq after ZNF829 perturbation: Assessing cell-type specific responses

When designing experiments to study ZNF829 in transcriptional networks, researchers should consider:

• The potential cooperative action with other zinc finger proteins

• Cell-type specificity of ZNF829 function

• Condition-dependent activity (e.g., stress response, developmental stage)

What are the methodological considerations for studying ZNF829 protein-protein interactions?

Investigating ZNF829's protein interaction network requires careful experimental design:

Immunoprecipitation-Based Methods:

• Standard co-IP protocol optimization:

  • Cell lysis conditions: Use gentle lysis buffers (e.g., 150 mM NaCl, 0.5% NP-40) to preserve interactions

  • Antibody selection: Choose antibodies (e.g., ABIN655374) targeting regions not involved in protein interactions

  • Washing stringency: Titrate salt concentration (150-300 mM) to balance specificity and sensitivity

• Crosslinking considerations:

  • Formaldehyde (0.1-1%): For capturing direct and indirect interactions

  • DSS or DSP (0.5-2 mM): For protein-protein crosslinking with reversibility

  • Optimization is crucial as excessive crosslinking may mask antibody epitopes

Advanced Interaction Proteomics:

• Proximity labeling protocols:

  • BioID-ZNF829 fusion expression followed by streptavidin pulldown

  • APEX2-ZNF829 with biotin-phenol labeling for temporal resolution

  • Compare labeled proteomes between wild-type and mutant ZNF829 to identify functional interaction domains

• Native protein complex isolation:

  • Size exclusion chromatography to isolate intact ZNF829 complexes

  • Blue native PAGE to preserve native complex structure

  • Ion exchange chromatography for fractionation based on charge properties

Data Analysis Framework:

• Prioritize interaction candidates based on:

  • Enrichment ratio compared to controls

  • Reproducibility across biological replicates

  • Known functions related to transcriptional regulation

  • Co-expression patterns with ZNF829

• Validation hierarchy:

  • Reciprocal co-IP with antibodies against interaction partners

  • Proximity ligation assay for in situ visualization

  • Functional validation through simultaneous perturbation

This methodological framework addresses the challenges of studying potentially transient or context-dependent interactions of transcription factors.

What are the current limitations in ZNF829 antibody-based research and potential alternative approaches?

Current ZNF829 antibody research faces several limitations that researchers should consider:

Technical Limitations:

• Antibody specificity concerns: Most available antibodies are polyclonal with potential batch-to-batch variation

• Limited epitope coverage: Available antibodies target specific regions, potentially missing important domains or isoforms

• Cross-reactivity with other zinc finger proteins: The zinc finger domain family has conserved structural elements

• Lack of monoclonal options: Few or no monoclonal antibodies are commercially available

Alternative Methodological Approaches:

Emerging Technologies with Potential Application:

• Single-molecule imaging: To visualize ZNF829 binding dynamics in real-time

• CUT&Tag/CUT&RUN: More sensitive alternatives to ChIP for mapping genomic binding sites

• Protein complementation assays: For studying specific protein-protein interactions

• CITE-seq: For simultaneous protein and RNA detection at single-cell resolution

When designing studies involving ZNF829, researchers should consider implementing multiple complementary approaches to overcome the limitations of antibody-based methods alone.

What are the recommended best practices for ensuring reproducibility in ZNF829 antibody-based experiments?

To maximize reproducibility in ZNF829 research:

Documentation and Reporting Standards:

• Comprehensive antibody information: Report catalog number, lot number, clone (if monoclonal), epitope region, and host species for all antibodies used

• Validation evidence: Document specificity validation performed in your experimental system

• Detailed methodological parameters: Include all buffer compositions, incubation times/temperatures, and equipment settings

• Image acquisition parameters: For microscopy and blot imaging, document exposure settings, gain, and any post-processing

Experimental Design Considerations:

• Independent antibody validation: Confirm results with at least two antibodies targeting different epitopes

• Appropriate controls: Include positive and negative controls specific to each application

• Biological replicates: Perform experiments across multiple independent biological samples

• Blinding and randomization: Implement where feasible to reduce unconscious bias

Reagent Authentication Procedures:

• Antibody performance testing: Establish quality control criteria before each new lot

• Cell line authentication: Regularly verify identity and mycoplasma status of cell models

• Recombinant protein standards: Use verified standards (e.g., NBP257673PEP) for quantification

A comprehensive reproducibility checklist tailored to ZNF829 research would include verification steps for each experimental technique, with specific criteria for acceptance of results and clear decision points for troubleshooting.

How might emerging single-cell technologies be applied to ZNF829 research?

Single-cell technologies offer unprecedented resolution for studying ZNF829 biology:

Single-Cell Genomic Applications:

• scRNA-seq with ZNF829 perturbation: Reveal cell type-specific transcriptional responses to ZNF829 modulation

• scATAC-seq: Map chromatin accessibility changes related to ZNF829 activity at single-cell resolution

• Single-cell ChIP-seq: Profile ZNF829 binding heterogeneity across individual cells

Spatial Transcriptomic Integration:

• MERFISH or seqFISH with ZNF829 immunostaining: Correlate ZNF829 protein levels with spatial transcriptomes

• Spatial proteomics: Map ZNF829 protein distribution in tissue context

• In situ sequencing: Detect ZNF829 binding sites in intact tissue sections

Multi-modal Profiling Strategies:

• CITE-seq adaptation: Add ZNF829 antibodies to ADT panels for simultaneous protein and RNA detection

• Combined genomic measurements: Integrate single-cell multiome approaches (RNA + ATAC, RNA + protein)

• Live cell imaging with endogenously tagged ZNF829: Track dynamics in combination with other labeled factors

Analytical Framework Development:

• Trajectory inference to map ZNF829 activity changes during cellular transitions

• Network analysis to identify cell type-specific ZNF829 regulatory networks

• Integration with existing single-cell atlases to contextualize findings

When implementing these approaches, researchers should consider:

• Antibody specificity and sensitivity at single-cell level

• Potential epitope masking in fixed cells

• Signal-to-noise challenges in low-abundance transcription factors

• Computational methods for integrating multi-modal data

What are the key unresolved questions in ZNF829 biology that require new methodological approaches?

Several fundamental questions about ZNF829 remain unanswered and may require innovative methodological solutions:

Knowledge Gaps and Methodological Challenges:

• DNA binding motif specificity:

  • Unresolved Question: What DNA sequences does ZNF829 recognize with high affinity?

  • Methodological Approach: Combine in vitro techniques (SELEX-seq, protein binding microarrays) with in vivo approaches (CUT&Tag, ChIP-exo) for high-resolution motif mapping

• Regulatory mechanisms controlling ZNF829 activity:

  • Unresolved Question: How is ZNF829 function regulated post-translationally?

  • Methodological Approach: Develop phospho-specific or modification-specific antibodies; apply mass spectrometry approaches to map PTM landscapes

• Biological contexts of ZNF829 function:

  • Unresolved Question: In which biological processes and cell types is ZNF829 most critical?

  • Methodological Approach: Conditional knockout models; single-cell multi-omics profiling across tissues and developmental stages

• Protein complex dynamics:

  • Unresolved Question: How do ZNF829 protein interactions change in response to cellular signals?

  • Methodological Approach: Time-resolved proximity labeling; synchronizable cellular systems with temporal proteomics

• Disease relevance:

  • Unresolved Question: Is ZNF829 dysregulation involved in human disease pathogenesis?

  • Methodological Approach: Analysis of disease-associated variants in ZNF829 binding sites; conditional expression in disease models

Emerging Technologies with Potential Impact:

• CRISPR epigenome editing: For targeted manipulation of ZNF829 binding sites

• Cryo-EM: For structural characterization of ZNF829 protein complexes

• Organoid models: For studying ZNF829 function in physiologically relevant 3D tissue contexts

• Patient-derived iPSCs: For investigating ZNF829 in human genetic disorders