ZFAND6 Antibody

What are the primary applications for ZFAND6 antibodies in research?

ZFAND6 antibodies are primarily used in Western blotting, immunofluorescence, immunohistochemistry (both paraffin and frozen sections), and enzyme-linked immunosorbent assays (ELISA). Western blotting represents the most commonly utilized application, with established protocols for detecting the approximately 22.6 kDa ZFAND6 protein .

For optimal results in Western blot applications, use dilutions between 1:50 and 1:400, adjusting based on antibody sensitivity and protein expression levels. For immunocytochemistry in formalin-fixed cells, dilutions of 1:50-1:500 are recommended, while immunohistochemistry applications typically require 1:50-1:500 for frozen sections and 1:10-1:100 for paraffin sections . For ELISA applications, dilutions of 1:100-1:200 are suggested, though optimization for specific experimental conditions is necessary .

What is known about ZFAND6 structure and localization?

Human ZFAND6 is a 208 amino acid protein with a molecular weight of approximately 22.6 kDa. The protein contains characteristic zinc finger domains, specifically the A20-like and AN1-type zinc finger domains, which are critical for its function in protein-protein interactions and ubiquitin binding .

ZFAND6 is predominantly localized in the cytoplasm under normal physiological conditions. Recent research has demonstrated that upon mitochondrial damage, ZFAND6 can be recruited to mitochondria, where it plays a crucial role in initiating mitophagy by mediating TRAF2-cIAP1 interactions at damaged mitochondrial sites . This dynamic localization is important when designing immunofluorescence experiments to track ZFAND6 redistribution under various cellular stresses.

How should ZFAND6 antibodies be stored for optimal performance?

For short-term storage, ZFAND6 antibodies can be stored at 4°C for frequent use. For long-term storage, store at -20°C to -80°C in a manual defrost freezer. Under proper storage conditions, antibodies can maintain activity for up to one year without detectable loss of function .

It is crucial to avoid repeated freeze-thaw cycles as these can lead to protein denaturation and loss of antibody activity. If frequent use is anticipated, consider aliquoting the antibody solution into smaller volumes before freezing to minimize freeze-thaw cycles. Most commercial ZFAND6 antibodies are supplied in PBS (pH 7.4) containing 0.02% NaN3 and 50% glycerol, which helps maintain stability during freezing .

What controls should be included when using ZFAND6 antibodies?

When designing experiments with ZFAND6 antibodies, several controls are essential:

• Positive control: Use recombinant ZFAND6 protein (such as Gly19~Ile208 with N-terminal His-Tag) or lysates from tissues known to express ZFAND6 highly (heart, skeletal muscle, liver, kidney, or placenta) .

• Negative control: Include ZFAND6 knockout or knockdown cells/tissues (if available) or samples known not to express ZFAND6.

• Antibody controls: Include primary antibody-only and secondary antibody-only controls to assess non-specific binding.

• Loading control: For Western blots, use housekeeping proteins (β-actin, GAPDH) to normalize for protein loading variations.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to confirm specificity of the signal.

Additionally, when investigating ZFAND6's role in mitophagy or TRAF2 interactions, include appropriate pathway controls such as mitochondrial markers (TOM20, COXIV) or TRAF2/cIAP1 detection .

How can I validate ZFAND6 antibody specificity for my research?

A multi-layered approach to validating ZFAND6 antibody specificity includes:

• Western blot analysis: Verify a single band of appropriate molecular weight (approximately 22.6 kDa for canonical ZFAND6). Be aware that up to two different isoforms have been reported .

• Genetic validation: Compare staining patterns between wild-type and ZFAND6 knockout/knockdown samples. Recent studies have successfully generated ZFAND6 knockout mice and cell lines that can serve as negative controls .

• Mass spectrometry: Confirm the identity of immunoprecipitated proteins using mass spectrometry.

• Orthogonal antibodies: Compare results using antibodies raised against different epitopes of ZFAND6.

• Recombinant expression: Overexpress tagged ZFAND6 and confirm co-detection with both anti-tag and anti-ZFAND6 antibodies.

For rigorous validation, combining at least three of these approaches is recommended, particularly when investigating novel functions of ZFAND6 such as its role in mitophagy or inflammatory regulation.

How can ZFAND6 antibodies be used to investigate mitophagy?

Recent research has revealed ZFAND6's critical role in mitophagy . To investigate this function:

• Co-localization studies: Use ZFAND6 antibodies in combination with mitochondrial markers (TOM20, COXIV) and autophagy markers (LC3, p62) in immunofluorescence to track recruitment to damaged mitochondria.

• Mitochondrial fractionation: Employ subcellular fractionation followed by Western blotting with ZFAND6 antibodies to quantify mitochondrial association under basal conditions versus after mitochondrial damage (e.g., CCCP treatment).

• Proximity ligation assay (PLA): Use ZFAND6 antibodies with antibodies against TRAF2 or cIAP1 to detect and quantify proximity-dependent interactions at mitochondria.

• Mitophagy flux assays: Combine ZFAND6 immunostaining with mitophagy reporters (mito-Keima, mito-QC) in the presence/absence of lysosomal inhibitors to assess mitophagy flux differences.

• Immunoprecipitation: Use ZFAND6 antibodies to pull down protein complexes after mitochondrial damage and identify interaction partners by mass spectrometry or Western blotting.

This multi-faceted approach can help elucidate how ZFAND6 bridges TRAF2-cIAP1 interactions during mitophagy and how its absence leads to accumulated damaged mitochondria and increased ROS .

What experimental approaches can resolve the paradox between in vitro viral resistance and in vivo susceptibility in ZFAND6-deficient models?

Recent studies have revealed an intriguing paradox where ZFAND6-deficient cells show enhanced viral resistance in vitro but ZFAND6-knockout mice exhibit increased susceptibility to influenza virus infection . To investigate this paradox:

• Comparative transcriptomics: Perform RNA-Seq on wild-type and ZFAND6-deficient cells/tissues both in vitro and in vivo after viral challenge to identify differentially regulated pathways.

• Cell-type specific analyses: Use ZFAND6 antibodies for immunohistochemistry or flow cytometry to assess expression patterns across different immune cell populations in infected tissues.

• Temporal immunoblotting: Monitor ZFAND6, ISG proteins, and viral proteins at multiple timepoints post-infection using Western blotting.

• Chimeric mouse models: Generate bone marrow chimeras to distinguish between the roles of ZFAND6 in hematopoietic versus non-hematopoietic cells during infection.

• Ex vivo culture systems: Isolate primary cells from infected animals for ex vivo analysis of viral replication and interferon responses.

These approaches can help reconcile how the spontaneous ISG expression in ZFAND6-deficient cells (due to mtDNA release) contributes to viral resistance in vitro, while impaired myeloid cell activation and diminished type I IFN signaling lead to increased mortality in vivo .

How can I use ZFAND6 antibodies to study its interaction with the TRAF2-cIAP E3 ubiquitin ligase complex?

ZFAND6 has been identified as a subunit of the TRAF2-cIAP E3 ubiquitin ligase complex . To study these interactions:

• Co-immunoprecipitation (Co-IP): Use ZFAND6 antibodies to pull down native protein complexes, followed by Western blotting for TRAF2, cIAP1, and other potential interactors.

• Reciprocal Co-IP: Perform immunoprecipitation with TRAF2 or cIAP1 antibodies and blot for ZFAND6.

• Domain mapping: Create truncated ZFAND6 constructs lacking specific domains (A20-like ZF domain or AN1 ZF domain) and assess which regions are required for TRAF2-cIAP1 interactions.

• Ubiquitination assays: Combine ZFAND6 antibodies with ubiquitin antibodies to detect ubiquitinated substrates in the presence/absence of ZFAND6.

• Proximity-dependent biotin identification (BioID): Fuse ZFAND6 to a biotin ligase to identify nearby proteins in living cells, then validate with co-IP using ZFAND6 antibodies.

These techniques can help elucidate how ZFAND6 bridges TRAF2-cIAP1 interactions and facilitates ubiquitin-dependent mitophagy to maintain mitochondrial homeostasis .

How can I improve detection of endogenous ZFAND6 in tissues with low expression?

When working with tissues or cells where ZFAND6 is expressed at low levels:

• Signal amplification: Consider using tyramide signal amplification (TSA) or other signal enhancement techniques for immunohistochemistry and immunofluorescence.

• Protein enrichment: For Western blotting, increase protein loading or perform subcellular fractionation to concentrate compartments where ZFAND6 is localized.

• Epitope retrieval optimization: Test multiple antigen retrieval methods (heat-induced vs. enzymatic, different pH buffers) to maximize epitope accessibility.

• Blocking optimization: Test different blocking solutions (BSA, non-fat milk, normal serum) to reduce background while preserving specific signal.

• Extended antibody incubation: Consider overnight primary antibody incubation at 4°C to improve signal-to-noise ratio.

Additionally, ZFAND6 expression can be induced under certain conditions, such as in response to cellular stress or inflammation . Consider using appropriate stimuli (e.g., TNF-α treatment, mitochondrial stress inducers) to upregulate ZFAND6 expression for easier detection.

What are the key considerations when designing co-localization experiments with ZFAND6?

When planning co-localization studies with ZFAND6 antibodies:

• Antibody compatibility: Ensure primary antibodies are raised in different host species to avoid cross-reactivity during secondary antibody detection.

• Fixation optimization: Test different fixation methods (paraformaldehyde, methanol, acetone) as they can differentially affect epitope accessibility and subcellular structure preservation.

• Sequential staining: For challenging combinations, consider sequential staining protocols with intermediate fixation steps.

• Channel selection: Choose fluorophores with minimal spectral overlap and appropriate controls for bleed-through correction.

• Quantitative analysis: Use appropriate co-localization metrics (Pearson's coefficient, Manders' coefficient) and software tools for objective assessment.

For mitochondrial co-localization specifically, induce mitochondrial damage (e.g., CCCP treatment) to enhance ZFAND6 recruitment to mitochondria before fixation, as recent research shows ZFAND6 is recruited to damaged mitochondria during mitophagy .

What are the implications of ZFAND6's role in mitochondrial quality control for disease research?

Recent discoveries regarding ZFAND6's function in mitophagy have significant implications for disease research:

• Neurodegenerative diseases: Given that impaired mitophagy is associated with conditions like Parkinson's disease, ZFAND6 dysfunction could contribute to neurodegeneration through accumulated damaged mitochondria and increased oxidative stress .

• Inflammatory disorders: ZFAND6 deficiency leads to cytosolic mtDNA release and spontaneous interferon responses, suggesting a potential role in inflammatory diseases characterized by type I interferon signatures .

• Metabolic disorders: ZFAND6 has been implicated as a potential susceptibility locus for type 2 diabetes in GWAS studies, which could be linked to its role in mitochondrial homeostasis and ROS regulation .

• Viral infections: The paradoxical findings regarding ZFAND6's role in viral defense suggest complex regulatory mechanisms that could inform new approaches to treating viral infections .

Research using ZFAND6 antibodies to examine protein expression, localization, and interactions in disease models could reveal new therapeutic targets for conditions associated with mitochondrial dysfunction and dysregulated inflammation.

How does ZFAND6 compare to other A20 family members in structure and function?

ZFAND6 belongs to the A20 family of proteins characterized by their zinc finger domains. Comparative analysis shows:

While A20 (TNFAIP3) primarily functions as a negative regulator of NF-κB signaling through its deubiquitinating activity, ZFAND6 appears to have evolved a specialized role in maintaining mitochondrial homeostasis through promoting mitophagy . When designing experiments with ZFAND6 antibodies, researchers should be aware of potential cross-reactivity with other A20 family members and include appropriate specificity controls.

Future research could explore the functional redundancy or cooperation between ZFAND6 and other A20 family members in regulating cellular stress responses and inflammation.