ZNF821 Antibody

What is ZNF821 and why is it significant in molecular biology research?

ZNF821 (Zinc Finger Protein 821) belongs to the C2H2-type zinc finger protein family, characterized by multiple zinc finger domains that facilitate DNA binding and potential transcriptional regulation. At least three isoforms of ZNF821 are known to exist, suggesting diverse functional roles in cellular processes . While less extensively characterized than some zinc finger proteins like ZNF827 (which has been identified as a single-stranded DNA binding protein involved in the ATR-CHK1 DNA damage response pathway), ZNF821 represents an important research target for understanding gene regulation mechanisms and potentially identifying novel therapeutic targets.

The significance of studying ZNF821 lies in expanding our understanding of zinc finger protein functions, which broadly include:

• Transcriptional regulation

• Chromatin modification

• Protein-protein interactions

• DNA damage response pathways

• Cell cycle control

What applications are most suitable for ZNF821 antibodies?

ZNF821 antibodies have been validated for multiple applications in molecular and cellular biology research:

Application suitability depends on the specific antibody characteristics, including epitope recognition, clonality, and species reactivity. Researchers should validate each antibody in their experimental system before proceeding with critical experiments.

How do ZNF821 isoforms differ, and which can current antibodies detect?

At least three isoforms of ZNF821 have been identified, though their structural and functional differences remain incompletely characterized in the literature . When selecting antibodies, researchers must consider which isoforms are relevant to their studies:

• Antibodies targeting amino acids 270-320 (like ABIN5690328) can detect the two largest isoforms of ZNF821 but may not recognize smaller variants lacking this region .

• Antibodies recognizing different epitopes (like HPA036372, which targets a region containing the sequence TNPNKVHCDSEGDEEETTQDEVSSHTSEEDGGVVKVEKELENTEQPVGGNEVVEHEVTGNLNSDPLLELCQCPLCQ) may have different isoform detection profiles .

Experimental validation using positive controls with known isoform expression patterns is recommended to confirm which isoforms a specific antibody can detect in your experimental system.

How can I validate the specificity of a ZNF821 antibody?

Rigorous validation is essential for ensuring reliable results with ZNF821 antibodies. Drawing from approaches used with related zinc finger proteins, researchers should implement multiple validation strategies:

• Genetic validation approaches:

  • siRNA/shRNA knockdown of ZNF821 followed by Western blot analysis

  • CRISPR-Cas9 knockout controls

  • Overexpression of tagged ZNF821 constructs

• Biochemical validation approaches:

  • Super-shift assays (if ZNF821 binds DNA, similar to approaches used for ZNF827)

  • Immunoprecipitation followed by mass spectrometry

  • Peptide competition assays using the immunizing peptide

• Imaging validation approaches:

  • Co-localization with known nuclear markers

  • Comparison of staining patterns across multiple antibodies targeting different ZNF821 epitopes

A comprehensive validation approach employing multiple techniques provides the strongest evidence for antibody specificity. For example, combining a super-shift assay, which demonstrated specificity for ZNF827 , with siRNA knockdown would provide robust validation for ZNF821 antibodies.

What experimental factors affect ZNF821 detection in immunofluorescence studies?

Optimizing ZNF821 detection by immunofluorescence requires careful consideration of multiple parameters:

Drawing from studies with related proteins like ZNF827, researchers may find that optimization of nuclear permeabilization is particularly important, as ZNF827 shows distinct nuclear localization patterns that co-localize with DNA damage markers . Similar patterns might be expected for ZNF821.

How do DNA-protein interactions impact ZNF821 antibody applications?

Zinc finger proteins often interact with DNA, which can affect antibody accessibility and experimental outcomes. Based on findings with the related protein ZNF827 , researchers should consider:

• Nuclease treatment effects:

  • In co-immunoprecipitation experiments with ZNF827, treatment with benzonase (a nuclease) disrupted interactions with RPA32 and RPA70, indicating DNA-mediated rather than direct protein interactions .

  • Similar considerations may apply to ZNF821 protein interaction studies.

• DNA-protein complex detection:

  • If ZNF821 forms stable complexes with DNA, antibody epitope accessibility may be affected.

  • Super-shift assays, as used with ZNF827 , can help determine if antibodies can recognize DNA-bound ZNF821.

• Chromatin association:

  • Chromatin immunoprecipitation (ChIP) applications may require specialized protocols optimized for zinc finger proteins.

  • Fixation conditions may need modification to preserve DNA-protein interactions while maintaining epitope accessibility.

Understanding and accounting for these DNA-protein interactions will improve experimental design and interpretation when working with ZNF821 antibodies.

What is the optimal protocol for ZNF821 antibody usage in Western blotting?

The following protocol is recommended for Western blot detection of ZNF821, adapted from general practices for nuclear proteins and specific information for ZNF821 antibodies:

Sample preparation:

• Lyse cells in RIPA buffer containing protease inhibitors

• Include benzonase treatment if DNA-protein interactions may affect extraction

• Quantify protein using Bradford or BCA assay

SDS-PAGE and transfer:

• Denature samples in Laemmli buffer (95°C, 5 minutes)

• Separate 20-40 μg protein/lane on 8-10% polyacrylamide gels

• Transfer to PVDF membrane (recommended for nuclear proteins)

Immunoblotting:

• Block membrane in 5% non-fat milk or BSA in TBST (1 hour, room temperature)

• Incubate with primary ZNF821 antibody at manufacturer's recommended dilution (overnight, 4°C)

• Wash 3× with TBST (10 minutes each)

• Incubate with HRP-conjugated secondary antibody (1 hour, room temperature)

• Wash 3× with TBST (10 minutes each)

• Develop using enhanced chemiluminescence

Technical considerations:

• Include positive controls (cell lines known to express ZNF821)

• Include molecular weight markers to identify isoforms (ZNF821 antibodies may detect multiple isoforms )

• For phosphorylation studies, use phosphatase inhibitors in lysis buffer

How can I optimize co-immunoprecipitation studies with ZNF821 antibodies?

Co-immunoprecipitation (co-IP) is valuable for studying ZNF821 protein interactions. The following protocol incorporates insights from studies of related zinc finger proteins:

Lysis and pre-clearing:

• Harvest cells and lyse in non-denaturing buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate with protease inhibitors)

• Clear lysate by centrifugation (14,000 × g, 10 minutes, 4°C)

• Pre-clear with Protein A/G beads (1 hour, 4°C)

Immunoprecipitation:

• Add 2-5 μg ZNF821 antibody to 500 μg-1 mg pre-cleared lysate

• Incubate overnight at 4°C with gentle rotation

• Add Protein A/G beads and incubate 2-4 hours at 4°C

• Wash beads 4× with lysis buffer

• Elute bound proteins by boiling in Laemmli buffer

Critical controls:

• Input sample (5-10% of lysate used for IP)

• IgG control (same species as ZNF821 antibody)

• Benzonase treatment control to distinguish DNA-mediated from direct protein interactions

Analytical considerations:

• To detect potentially transient interactions, consider crosslinking before lysis

• For studying DNA-dependent interactions, compare results with and without nuclease treatment

• For mass spectrometry analysis, consider more gentle elution methods

What approaches enable reliable detection of ZNF821 in fixed tissue sections?

Immunohistochemical detection of ZNF821 in tissues requires careful protocol optimization:

Tissue preparation:

• Fix tissues in 10% neutral buffered formalin or 4% paraformaldehyde

• Process and embed in paraffin following standard protocols

• Section at 4-6 μm thickness

Antigen retrieval and staining:

• Deparaffinize and rehydrate sections

• Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Block endogenous peroxidase activity with 3% H₂O₂

• Block non-specific binding with 5-10% normal serum

• Incubate with ZNF821 antibody at 1:200-1:500 dilution (overnight, 4°C)

• Wash 3× with PBS or TBS

• Apply appropriate detection system (e.g., polymer-HRP)

• Develop with DAB or other chromogen

• Counterstain, dehydrate, and mount

Validation approaches:

• Include positive control tissues

• Include absorption controls (pre-incubation of antibody with immunizing peptide)

• Compare patterns across multiple ZNF821 antibodies targeting different epitopes

For fluorescent detection, substitute the chromogenic steps with fluorophore-conjugated secondary antibodies and include appropriate counterstains (e.g., DAPI for nuclei).

How can I perform multicolor immunofluorescence with ZNF821 antibodies?

Multicolor immunofluorescence allows co-localization analysis of ZNF821 with other proteins of interest:

Protocol:

• Fix cells using 4% paraformaldehyde (10 minutes, room temperature)

• Permeabilize with 0.2% Triton X-100 (10 minutes)

• Block with 5% normal serum from secondary antibody species

• Co-incubate with ZNF821 antibody (0.25-2 μg/mL) and antibodies against proteins of interest

• Wash 3× with PBS

• Incubate with species-specific secondary antibodies with non-overlapping fluorescence spectra

• Wash 3× with PBS

• Counterstain nuclei with DAPI

• Mount in anti-fade medium

Technical considerations:

• If primary antibodies are from the same species, use directly labeled antibodies or sequential staining protocols

• For co-localization analysis, acquire images using sequential scanning to prevent bleed-through

• Include single-label controls to confirm specificity and absence of cross-reactivity

• Consider super-resolution microscopy for detailed co-localization studies

This approach has been successfully used to demonstrate co-localization of ZNF827 with both γH2AX and RPA32 at DNA damage sites , and similar approaches can be adapted for ZNF821 studies.

How can I troubleshoot non-specific binding with ZNF821 antibodies?

Non-specific binding can compromise experimental results. The following troubleshooting strategies address common issues:

For nuclear proteins like ZNF821, particular attention should be paid to nuclear extraction efficiency and permeabilization conditions in immunofluorescence applications.

What controls are essential for validating ZNF821 antibody experiments?

Robust experimental design includes appropriate controls to ensure data validity:

Western blotting controls:

• Positive control (cell line known to express ZNF821)

• Negative control (cell line with low/no ZNF821 expression)

• Loading control (housekeeping protein)

• siRNA/shRNA knockdown control

• Peptide competition control

Immunofluorescence/IHC controls:

• Primary antibody omission control

• Isotype control (irrelevant antibody of same isotype and concentration)

• Peptide competition control

• Known positive tissue/cell control

• Subcellular marker co-staining (e.g., nuclear marker)

IP/Co-IP controls:

• Input sample (5-10% of lysate)

• IgG control

• Nuclease treatment control to distinguish DNA-mediated interactions

• Reverse IP validation (IP with antibody against interacting protein)

These controls help distinguish specific signals from artifacts and ensure reliable interpretation of experimental results.

How can ZNF821 antibodies be used to study protein-DNA interactions?

Given the nature of zinc finger proteins as DNA-binding factors, studying ZNF821-DNA interactions is of significant interest. Several methodologies can be adapted from approaches used with related proteins:

Chromatin Immunoprecipitation (ChIP):

• Cross-link protein-DNA complexes with formaldehyde

• Sonicate chromatin to 200-500 bp fragments

• Immunoprecipitate with ZNF821 antibody

• Reverse cross-links and purify DNA

• Analyze by qPCR, sequencing, or microarray

Electrophoretic Mobility Shift Assay (EMSA) with supershift:

• Incubate nuclear extracts with labeled DNA probe

• Add ZNF821 antibody to induce supershift

• Resolve complexes by native PAGE

• Verify specificity with competitive unlabeled probe

Drawing from studies of ZNF827, which demonstrated DNA binding through EMSA and antibody supershift assays , similar approaches can be applied to characterize ZNF821 DNA binding properties.

What methods allow investigation of ZNF821 in the context of DNA damage response?

If ZNF821 functions similarly to ZNF827 in DNA damage response , several methodologies can be employed:

Laser micro-irradiation coupled with live imaging:

• Express fluorescently-tagged ZNF821 in cells

• Perform laser micro-irradiation to induce localized DNA damage

• Monitor recruitment kinetics of ZNF821 to damage sites

• Co-visualize with known DNA damage markers

Immunofluorescence after DNA damage induction:

• Treat cells with DNA damaging agents (e.g., hydroxyurea, camptothecin)

• Fix and stain for ZNF821 and DNA damage markers

• Quantify colocalization and recruitment dynamics

Functional assays:

• Deplete ZNF821 using siRNA or CRISPR-Cas9

• Assess impact on DNA damage signaling (e.g., γH2AX, pCHK1)

• Measure DNA repair efficiency using reporter assays

• Evaluate cellular sensitivity to DNA damaging agents

These approaches have successfully revealed ZNF827's role in ATR-CHK1 pathway activation and could uncover similar functions for ZNF821.

What are the current knowledge gaps regarding ZNF821 antibody applications?

Despite available antibodies and methodologies, several knowledge gaps remain:

• Isoform-specific detection: While we know at least three isoforms of ZNF821 exist , comprehensive characterization of isoform-specific antibodies is lacking.

• Functional domains: Unlike ZNF827, whose zinc finger domains have been functionally characterized for DNA binding , the specific functional domains of ZNF821 require further investigation.

• Post-translational modifications: Antibodies specifically recognizing modified forms of ZNF821 (phosphorylated, ubiquitinated, etc.) would enhance our understanding of its regulation.

• Tissue-specific expression: Comprehensive immunohistochemical analysis across diverse tissues would clarify ZNF821's expression patterns and potential functions.

• Interactome characterization: Antibodies suitable for immunoprecipitation-mass spectrometry approaches would facilitate mapping of the ZNF821 protein interaction network.