chchd6a Antibody

What is CHCHD6A and what are its primary cellular functions?

CHCHD6A is a member of a family of proteins containing a conserved (coiled coil 1)-(helix 1)-(coiled coil 2)-(helix 2) domain. It functions as a component of the MICOS complex (mitochondrial contact site and cristae organizing system) located in the inner membrane of mitochondria . This protein plays essential roles in:

• Maintaining mitochondrial cristae morphology

• Regulating oxidative phosphorylation

• Supporting mitochondrial dynamics and ATP production

• Interacting with multiple mitochondrial proteins including mitofilin, SAM50, metaxins 1 and 2, and CHCHD3

Research has also shown that CHCHD6 maintains cristae integrity through interaction with TMEM65, a mitochondrial membrane protein. Importantly, CHCHD6 demonstrates dual localization capabilities, showing nuclear translocation under cellular stress conditions where it modulates transcription factors such as NFE2L2 to influence antioxidant responses.

How are CHCHD6A antibodies characterized and validated?

CHCHD6A antibodies require rigorous validation through multiple complementary techniques:

Robust validation includes testing against known positive controls and analyzing knockout or knockdown samples to confirm specificity. Some antibodies, like those developed against human CHCHD6, have been validated through the NIH's Antibody Characterization Lab initiatives.

What are key differences between zebrafish CHCHD6A and human CHCHD6 antibodies?

When selecting between zebrafish-specific and human CHCHD6 antibodies, researchers should consider several important distinctions:

• Species reactivity: Zebrafish CHCHD6A antibodies (e.g., CSB-PA714459XA01DIL) are specifically developed against recombinant Danio rerio CHCHD6A protein and may not cross-react with mammalian models .

• Molecular identification: Zebrafish CHCHD6A corresponds to UniProt number Q63ZW2, Entrez Gene ID 449542, and KEGG dre:449542 .

• Validation components: Some zebrafish-specific antibody kits include antigens (200μg) for positive control and pre-immune serum for negative control, enabling thorough validation .

• Application range: Human CHCHD6 antibodies typically offer broader application potential across Western blot, IHC, ICC, IF, IP, and ELISA with established protocols .

How does CHCHD6 function in cancer biology and what implications does this have for research?

CHCHD6 demonstrates significant roles in cancer progression and treatment response:

• Overexpression patterns: CHCHD6 overexpression correlates with poor prognosis in multiple cancer types, including glioblastoma and breast cancer.

• Therapeutic potential: CHCHD6 knockdown in human cancer cells enhances their sensitivity to genotoxic anticancer drugs, suggesting potential as a therapeutic target .

• Mechanistic pathway: In colorectal cancer models, CHCHD6 depletion disrupts mitochondrial dynamics, impairing EGF/Wnt signaling pathways and tumor progression.

For cancer researchers, CHCHD6A antibodies are valuable tools for:

• Detecting CHCHD6 in tumor biopsies to assess mitochondrial dysfunction

• Screening compounds targeting MICOS complex proteins

• Evaluating the relationship between mitochondrial integrity and cancer cell metabolism

Additionally, the relationship between CHCHD6 and cell signaling pathways offers research opportunities at the intersection of mitochondrial function and oncogenic signaling.

What experimental considerations are important when studying CHCHD6A's role in mitochondrial dynamics?

When designing experiments to investigate CHCHD6A's impact on mitochondrial dynamics, researchers should consider:

• Functional readouts: CHCHD6 knockdown causes measurable reductions in:

  • Oxygen consumption rate (OCR)

  • ATP production

  • Mitochondrial mass (quantifiable via flow cytometry with MitoTracker Red staining)

• Morphological analysis: Appropriate techniques include:

  • Electron microscopy to evaluate cristae morphology

  • Super-resolution microscopy to visualize MICOS complexes

  • Co-localization studies with other mitochondrial markers

• Protein interaction network:

  • Design co-immunoprecipitation experiments to capture interactions with known partners (mitofilin, SAM50, metaxins 1 and 2, CHCHD3)

  • Include appropriate detergent conditions to maintain membrane protein interactions

• Functional compensation: Consider potential compensatory mechanisms by related proteins, particularly other CHCHD family members that may obscure phenotypes in knockdown studies.

How can CHCHD6A antibodies be utilized in studying the MICOS complex architecture?

The MICOS complex represents a sophisticated protein assembly critical for mitochondrial structure. CHCHD6A antibodies provide valuable tools for dissecting this complex:

• Stoichiometry determination: Use quantitative immunoblotting with CHCHD6A antibodies alongside antibodies against other MICOS components to establish relative protein ratios.

• Assembly dynamics: Employ pulse-chase experiments combined with immunoprecipitation to track the temporal incorporation of CHCHD6A into the MICOS complex.

• Structural interactions: Utilize proximity ligation assays (PLA) with CHCHD6A antibodies paired with antibodies against proposed interaction partners.

• Localization precision: Implement super-resolution microscopy techniques (STORM, PALM) using fluorescently-labeled CHCHD6A antibodies to map nanoscale distribution within mitochondria.

For optimal results, researchers should consider using epitope-specific antibodies that target distinct domains of CHCHD6A, such as those recognizing aa 135-235 or aa 1-235 , to distinguish functional regions within the assembled complex.

What are the optimal conditions for Western blot analysis using CHCHD6A antibodies?

For successful Western blot detection of CHCHD6A, researchers should optimize several parameters:

When troubleshooting:

• Expected molecular weight of human CHCHD6 is approximately 26-29 kDa

• Include appropriate positive controls (e.g., HeLa lysates for human CHCHD6)

• Consider sample preparation modifications if working with membrane-associated fractions

How should researchers optimize immunohistochemistry protocols for CHCHD6A detection?

Successful immunohistochemical detection of CHCHD6A requires careful attention to tissue processing and staining conditions:

• Fixation optimization:

  • For paraffin-embedded sections: 10% neutral buffered formalin (24-48 hours)

  • For frozen sections: 4% paraformaldehyde (10-15 minutes)

  • Overfixation may mask epitopes; consider optimization experiments

• Antigen retrieval methods:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimization may be required depending on tissue type and fixation duration

• Antibody dilution and incubation:

  • Starting dilution: 1:50-400 for IHC applications

  • Optimal incubation: overnight at 4°C in a humidified chamber

  • Include controls: (1) omit primary antibody, (2) use pre-immune serum, (3) use tissue with known expression patterns

• Signal development system:

  • DAB (3,3'-diaminobenzidine) for brightfield microscopy

  • Fluorophore-conjugated secondary antibodies for fluorescence microscopy

  • Consider tyramide signal amplification for low-abundance targets

• Counterstaining recommendations:

  • Hematoxylin for nuclear counterstain in DAB-based detection

  • DAPI for nuclear visualization in fluorescence-based methods

What strategies can improve immunoprecipitation success when using CHCHD6A antibodies?

Immunoprecipitation of CHCHD6A presents challenges due to its membrane association and protein complex participation. Consider these approaches:

• Lysis buffer optimization:

  • Use mild detergents (0.5-1% NP-40 or Triton X-100) to maintain protein interactions

  • Include protease inhibitors and phosphatase inhibitors if studying post-translational modifications

  • Consider digitonin (0.5-1%) for preserving membrane protein complexes

• Antibody selection and coupling:

  • Use affinity-purified antibodies with demonstrated IP capability

  • Consider covalent coupling to protein A/G beads to prevent antibody contamination in eluted samples

  • Optimize antibody:lysate ratio through titration experiments

• Pre-clearing strategy:

  • Implement thorough pre-clearing with protein A/G beads

  • Consider including non-immune IgG from the same species as the CHCHD6A antibody

• Elution conditions:

  • Use gentle elution with antibody-specific peptide for native conditions

  • Apply more stringent SDS-based elution for complete recovery

• Verification approaches:

  • Confirm successful IP by Western blot

  • Consider mass spectrometry to identify co-immunoprecipitated proteins

  • Use reciprocal IP with known interaction partners to validate findings

How can researchers effectively design CHCHD6A knockdown experiments?

When designing CHCHD6A knockdown studies, researchers should implement comprehensive strategies to ensure robust phenotypic analysis:

• Knockdown approach selection:

  • siRNA for transient effects (evaluate 48-72 hours post-transfection)

  • shRNA for stable knockdown in long-term studies

  • CRISPR-Cas9 for complete knockout when appropriate

• Validation requirements:

  • Confirm knockdown efficiency by both qRT-PCR and Western blot

  • Expected phenotypes include defects in mitochondrial cristae morphology and reductions in cell growth, ATP production, and oxygen consumption

  • Monitor mitofilin protein levels, which typically decrease following CHCHD6 knockdown

• Essential controls:

  • Non-targeting siRNA/shRNA

  • Rescue experiments with siRNA/shRNA-resistant CHCHD6A constructs

  • Phenotypic comparison with knockdown of interaction partners (e.g., mitofilin)

• Phenotypic analysis panel:

  • Mitochondrial morphology (fluorescence microscopy with MitoTracker)

  • Functional assays (oxygen consumption, ATP production)

  • Cell growth and proliferation measurements

  • For cancer studies: sensitivity to genotoxic agents

What factors should be considered when selecting between different CHCHD6A antibody epitopes?

The choice of antibody epitope can significantly impact experimental outcomes:

When selecting between epitopes, researchers should consider:

• The structural accessibility of the epitope in native vs. denatured conditions

• Whether post-translational modifications may affect epitope recognition

• The conservation of the epitope sequence across species if cross-reactivity is desired

• The proximity of the epitope to functional domains or interaction interfaces

How should researchers interpret contradictory results between different detection methods when studying CHCHD6A?

When faced with discrepancies between different detection methods for CHCHD6A, consider these systematic troubleshooting approaches:

• Method-specific considerations:

  • Western blot may not detect certain post-translational modifications

  • IHC/IF may show different patterns due to epitope masking in fixed tissues

  • RNA expression (qPCR) may not correlate with protein levels due to post-transcriptional regulation

• Reconciliation strategies:

  • Validate with multiple antibodies targeting different epitopes

  • Employ genetic approaches (knockdown/knockout) as definitive controls

  • Consider cell type-specific or condition-dependent expression patterns

• Common sources of discrepancy:

  • Nuclear vs. mitochondrial localization depending on cellular stress

  • Interspecies variation in expression or epitope sequence

  • Interaction-dependent epitope masking

  • Technical variables including fixation methods, detergent compatibility, and antibody clone specificity

• Resolution approaches:

  • Subcellular fractionation to distinguish compartment-specific expression

  • Mass spectrometry validation of protein identity

  • Alternative techniques such as proximity ligation assay to validate interactions

  • Careful assessment of positive and negative controls across all methods

How might CHCHD6A antibodies contribute to emerging research on mitochondrial-nuclear communication?

CHCHD6's dual localization capability positions it as a potential mediator in mitochondrial-nuclear crosstalk:

• Stress-dependent translocation studies:

  • CHCHD6A antibodies can track nuclear translocation under various cellular stressors

  • Immunofluorescence co-localization with nuclear markers can quantify translocation dynamics

  • ChIP-seq approaches using CHCHD6A antibodies may identify genomic binding sites

• Transcriptional regulation:

  • CHCHD6 modulates transcription factors like NFE2L2 (Nrf2)

  • CHCHD6A antibodies can be employed in promoter-binding studies

  • Sequential ChIP (Re-ChIP) can identify co-regulatory complexes

• Retrograde signaling mechanisms:

  • Investigate CHCHD6A's role in communicating mitochondrial status to the nucleus

  • Examine post-translational modifications that might regulate localization

  • Study interaction partners in both compartments using co-immunoprecipitation with CHCHD6A antibodies

This research area presents opportunities to understand how mitochondrial proteins may directly influence nuclear gene expression during cellular adaptation to stress.

What role might CHCHD6A play in therapeutic resistance, and how can antibodies facilitate this research?

Based on current evidence of CHCHD6's involvement in cancer progression, several research avenues emerge:

• Predictive biomarker potential:

  • CHCHD6A antibodies can assess expression levels in patient samples

  • Correlation studies between CHCHD6 levels and treatment response

  • Development of standardized IHC protocols for clinical evaluation

• Mechanistic investigations:

  • CHCHD6 knockdown enhances sensitivity to genotoxic anticancer drugs

  • Antibodies can monitor expression changes during treatment resistance development

  • Co-immunoprecipitation studies to identify resistance-specific interaction partners

• Combination therapy approaches:

  • Studies indicate that targeting the CHD6-TMEM65 axis may be effective for cancer intervention

  • Antibodies against both proteins can monitor relationship dynamics during treatment

  • Evaluate potential for synergistic effects with existing therapies

• Therapeutic target validation:

  • CHCHD6A antibodies can confirm target engagement of novel therapeutics

  • Monitor on-target vs. off-target effects through immunohistochemistry

  • Assess pathway modulation in response to targeted therapies

Researchers investigating CHCHD6A in therapeutic contexts should consider implementing tissue microarray analysis with standardized antibody protocols to facilitate larger cohort studies.