CHCHD6 Antibody

What is CHCHD6 and why is it important in mitochondrial research?

CHCHD6 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 core component of the mitochondrial contact site and cristae organizing system (MICOS). CHCHD6 has been observed in a complex with other mitochondrial proteins including mitofilin, SAM50, metaxins 1 and 2, and CHCHD3 . This protein is critical for maintaining proper mitochondrial cristae morphology, which directly impacts cellular bioenergetics. CHCHD6 knockdown leads to severe defects in mitochondrial cristae structure and causes reductions in cell growth, ATP production, and oxygen consumption . Recent research has also identified CHCHD6 as a potential therapeutic target in cancer, as its knockdown enhances cancer cell sensitivity to genotoxic anticancer drugs .

What applications are CHCHD6 antibodies typically used for?

CHCHD6 antibodies are employed in multiple experimental approaches:

The antibody choice should be based on the specific experimental needs, with polyclonal antibodies offering broader epitope recognition and monoclonal antibodies providing higher specificity for particular epitopes .

How should CHCHD6 antibodies be stored and handled?

For optimal performance, CHCHD6 antibodies should be stored at -20°C and remain stable for approximately one year after shipment. Many commercial CHCHD6 antibodies are supplied in PBS containing 0.02% sodium azide and 50% glycerol at pH 7.3 . Small aliquots (20μl) may contain 0.1% BSA as a stabilizer . Avoid repeated freeze-thaw cycles as this can compromise antibody functionality. When handling the antibody, maintain sterile conditions and use appropriate personal protective equipment due to the presence of sodium azide, which is toxic and can form explosive compounds with heavy metals in plumbing systems.

What are the optimal conditions for Western blot detection of CHCHD6?

When performing Western blot for CHCHD6 detection, researchers should consider these methodological aspects:

• Expected Molecular Weight: The calculated molecular weight of CHCHD6 is 26 kDa (235 amino acids), but it typically appears between 26-29 kDa on SDS-PAGE gels .

• Sample Preparation: Total cell lysates or mitochondrial fractions can be used. For mitochondrial enrichment, consider using differential centrifugation protocols.

• Blocking Solution: 5% non-fat milk in TBST is generally effective.

• Primary Antibody Dilution: Start with 1:1000-1:4000 dilution and optimize based on signal strength .

• Positive Controls: A431, HeLa, or Jurkat cell lysates are recommended as positive controls .

• Detection Method: Both chemiluminescence and fluorescence-based detection methods work well for CHCHD6.

The specificity of the antibody should be confirmed using knockout or knockdown controls, as demonstrated in studies using TALEN-mediated knockdown of CHCHD6 .

How can I effectively use CHCHD6 antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) is valuable for studying CHCHD6 interactions with other mitochondrial proteins. For optimal results:

• Lysis Buffer Selection: Use mild non-denaturing buffers (e.g., NP-40 or Triton X-100 based) to preserve protein-protein interactions.

• Antibody Amount: Use 0.5-4.0 μg of CHCHD6 antibody per 1.0-3.0 mg of total protein lysate .

• Pre-clearing: Pre-clear lysates with appropriate control IgG and protein A/G beads to reduce non-specific binding.

• Negative Controls: Include an IgG control from the same species as the CHCHD6 antibody.

• Validation: Confirm pulled-down complexes by immunoblotting for known interaction partners (mitofilin, Sam50, CHCHD3, OPA1) .

Research has shown that CHCHD6 co-immunoprecipitates with mitofilin, Sam50, and CHCHD3, forming part of the MICOS complex . Additionally, CHCHD6 has been shown to interact with the soluble form of OPA1 but not the long form .

What methodologies are recommended for studying CHCHD6 localization?

For accurate subcellular localization of CHCHD6:

• Immunofluorescence Protocol:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.2% Triton X-100

  • Block with 5% BSA or normal serum

  • Incubate with CHCHD6 antibody (1:200-1:800 dilution)

  • Co-stain with established mitochondrial markers (e.g., MitoTracker, TOMM20)

• Super-resolution Microscopy: Consider techniques like STED or STORM for detailed visualization of CHCHD6 within mitochondrial structures.

• Mitochondrial Fractionation: Separate submitochondrial fractions (outer membrane, inner membrane, intermembrane space, matrix) to determine precise localization using biochemical approaches.

• Proximity Labeling: Techniques like BioID or APEX2 fused to CHCHD6 can help identify proximal proteins and confirm localization within the mitochondrial architecture.

Given CHCHD6's role in cristae organization, correlative light and electron microscopy can provide valuable insights into its relationship with mitochondrial ultrastructure .

What are effective approaches for CHCHD6 knockdown or knockout in research models?

Several genetic manipulation strategies have been validated for CHCHD6:

• CRISPR-Cas9 System:

  • Successfully used to generate CHCHD6 knockout cell lines

  • Synthetic guide RNA (sgRNA) targeting CHCHD6 has been validated in HT-22 cells

  • Stable knockouts require puromycin selection (typically 5 μg/mL for selection, maintained at 2 μg/mL)

• TALENs (Transcription Activator-Like Effector Nucleases):

  • Effective for CHCHD6 gene targeting

  • Has been used to generate CHCHD6 knockout clones with homozygous frameshift mutations

  • Screening typically requires sequencing and immunoblot validation

• shRNA-Mediated Knockdown:

  • AAV-mCherry-CHCHD6 shRNA has been used for in vivo knockdown

  • Recommended viral concentration: 1.0e12 GC/mL (genome copies/mL)

  • Typically 1 μL injected per hemisphere in mouse models

For appropriate controls, both AAV-GFP-CHCHD6 (for overexpression) and AAV-mCherry-Scrambled shRNA (for knockdown controls) have been validated in research settings .

How can I validate CHCHD6 knockout or knockdown efficiency?

Thorough validation is essential when working with genetic manipulations:

• Genomic Verification:

  • PCR amplification and sequencing of the targeted region

  • T7 Endonuclease I assay for detection of mutations

• Protein Level Verification:

  • Western blot using validated CHCHD6 antibodies (dilution 1:1000-1:4000)

  • Immunofluorescence to confirm loss of signal in modified cells

• Functional Validation:

  • Assessment of mitochondrial morphology using transmission electron microscopy (TEM)

  • Measurement of cristae density and organization

  • Analysis of mitochondrial membrane potential (ΔΨm)

  • Quantification of intracellular ATP content

Research has shown that CHCHD6 knockout cells exhibit decreased cristae density, while still maintaining some mitochondrial function. Complete loss of Mitofilin, by contrast, leads to more severe vesicle-like cristae morphology changes and functional impairments .

How can CHCHD6 antibodies be used to investigate the relationship between mitochondrial dysfunction and neurodegenerative diseases?

Recent research has uncovered a crucial link between CHCHD6 and Alzheimer's disease (AD) pathology:

• CHCHD6-APP Axis Investigation:

  • CHCHD6 antibodies can be used to examine the physical interaction between CHCHD6 and amyloid-beta precursor protein (APP)

  • Co-immunoprecipitation assays reveal that APP and CHCHD6 bind and stabilize one another under normal conditions

  • Western blot analysis shows decreased CHCHD6 levels in AD patient brains and AD model systems

• Transcriptional Regulation Analysis:

  • ChIP assays using CHCHD6 promoter-specific primers can confirm binding of APP intracellular domain (AICD) to the CHCHD6 promoter

  • CHCHD6 is transcriptionally down-regulated by AICD binding to its promoter, with Fe65 and Tip60 serving as cofactors

• Mitochondria-Associated ER Membranes (MAM) Studies:

  • Subcellular fractionation combined with CHCHD6 antibody detection can examine how CHCHD6 loss affects APP accumulation on MAM

  • This methodology has revealed that CHCHD6 depletion promotes APP accumulation on MAM, accelerating aberrant APP processing

These approaches have demonstrated that compensation for CHCHD6 loss in AD mouse models can reduce AD-associated neuropathology and cognitive impairment, suggesting therapeutic potential .

What methods can be used to study the interaction between CHCHD6 and other MICOS complex components?

Understanding CHCHD6's role within the MICOS complex requires sophisticated interaction analyses:

• Sequential Co-Immunoprecipitation:

  • First immunoprecipitate with CHCHD6 antibody

  • Analyze precipitates for mitofilin, Sam50, CHCHD3, and other MICOS components

  • This approach has confirmed a complex containing Mitofilin, Sam50, and CHCHD3/6

• Domain Mapping:

  • Generate truncation mutants of CHCHD6 to identify interaction domains

  • Research has shown CHCHD6 binds to Sam50 through its N-terminus, similar to CHCHD3

  • The CHCH domain appears important for Mitofilin interaction

• Crosslinking Mass Spectrometry:

  • Chemical crosslinking followed by mass spectrometry can identify precise interaction sites

  • Helps distinguish direct from indirect interactions within the complex

• Proximity-Dependent Biotin Identification (BioID):

  • Fusion of BioID or TurboID to CHCHD6 allows identification of proximal proteins

  • Provides spatial information about the arrangement of MICOS components

These methods have revealed that CHCHD6, while sharing 36% sequence identity with CHCHD3, has both overlapping and distinct functions within the MICOS complex .

How can transmission electron microscopy be used in conjunction with CHCHD6 antibodies to study cristae morphology?

Correlating CHCHD6 levels with cristae structure requires integrated approaches:

These techniques have established that CHCHD6 is essential for maintaining normal cristae structure, with its loss causing distinct morphological alterations that impact mitochondrial function .

Why might I observe inconsistent results when using CHCHD6 antibodies in different cell types?

Several factors can contribute to variability in CHCHD6 antibody performance across cell types:

• Expression Level Differences:

  • CHCHD6 expression varies naturally between cell types

  • Confirm baseline expression through qPCR before antibody-based experiments

  • Adjust antibody concentration based on expected expression levels

• Post-Translational Modifications:

  • Cell-specific PTMs may affect epitope accessibility

  • Consider using multiple antibodies targeting different regions of CHCHD6

  • The observed molecular weight of CHCHD6 (26-29 kDa) suggests potential modifications

• Protein-Protein Interactions:

  • Cell type-specific interaction partners may mask epitopes

  • Try different lysis conditions to disrupt interactions

  • Consider native vs. denaturing conditions depending on experimental goals

• Mitochondrial Heterogeneity:

  • Mitochondrial content and morphology differ between cell types

  • Normalize to mitochondrial mass using markers like VDAC or TOM20

  • Consider cell-specific optimization of mitochondrial isolation procedures

When transitioning between different cell models, always re-optimize antibody concentrations and validation procedures rather than directly applying protocols established in another cell type.

What controls should I include when using CHCHD6 antibodies for experimental validation?

Rigorous controls are essential for reliable CHCHD6 antibody-based research:

• Positive Controls:

  • Cell lines with confirmed CHCHD6 expression: A431, HeLa, and Jurkat cells

  • Recombinant CHCHD6 protein (when available)

  • Overexpression systems (AAV-GFP-CHCHD6 has been validated)

• Negative Controls:

  • CHCHD6 knockout or knockdown models

  • CRISPR-Cas9 generated CHCHD6 knockout HT-22 cells

  • TALENs-generated CHCHD6 knockout clones with homozygous frameshift mutations

  • AAV-mCherry-CHCHD6 shRNA for knockdown approaches

• Specificity Controls:

  • Peptide competition assays with the immunizing peptide

  • Secondary antibody-only controls to assess non-specific binding

  • IgG controls matched to the host species and concentration

• Cross-Reactivity Controls:

  • Related proteins (especially CHCHD3, which shares 36% sequence identity)

  • Consider double knockout/knockdown approaches to address redundancy

These controls help distinguish true CHCHD6 signal from artifacts and provide confidence in experimental findings, particularly when investigating novel CHCHD6 functions or interactions.

How can CHCHD6 antibodies be utilized in investigating potential therapeutic approaches for neurodegenerative diseases?

Recent discoveries about the CHCHD6-APP axis open new therapeutic exploration avenues:

• Target Validation:

  • CHCHD6 antibodies can monitor protein levels before and after candidate therapeutic interventions

  • Verification of target engagement through proximity ligation assays between CHCHD6 and APP

  • Assessment of downstream effects on mitochondrial function and amyloid pathology

• Biomarker Development:

  • Potential use of CHCHD6 levels as a biomarker for mitochondrial dysfunction in AD

  • Correlation studies between CHCHD6 levels and disease progression

  • Development of sensitive ELISAs using validated CHCHD6 antibodies

• Therapeutic Response Monitoring:

  • CHCHD6 antibodies can evaluate the efficacy of CHCHD6-stabilizing compounds

  • Monitor restoration of CHCHD6-APP interactions following treatment

  • Assess normalization of mitochondrial cristae morphology using complementary EM techniques

• Animal Model Validation:

  • AAV-mediated CHCHD6 overexpression has shown promising results in reducing AD-associated neuropathology and cognitive impairment in mouse models

  • CHCHD6 antibodies are essential for validating protein levels after AAV delivery

  • Recommended viral dosage: 1.0e9 viral particles per hemisphere

This research direction highlights CHCHD6 stabilization as a novel therapeutic target with potential to address both amyloid pathology and mitochondrial dysfunction in AD .

What are the recommended approaches for studying CHCHD6's role in cancer research?

CHCHD6's potential as an anti-tumor target requires specialized experimental approaches:

• Cancer Cell Line Profiling:

  • Western blot analysis of CHCHD6 expression across cancer cell lines

  • Correlation with mitochondrial function parameters

  • CHCHD6 knockdown has been shown to enhance cancer cell sensitivity to genotoxic drugs

• Drug Sensitivity Assays:

  • Generate stable CHCHD6 knockdown cancer cell lines

  • Evaluate dose-response curves for various chemotherapeutics

  • Assess mechanisms of sensitization (apoptosis, necrosis, mitochondrial dysfunction)

• In Vivo Tumor Models:

  • Monitor tumor growth in xenograft models with modulated CHCHD6 expression

  • Assess therapeutic response using CHCHD6 antibody-based IHC

  • Evaluate mitochondrial cristae integrity in tumor samples

• Patient Sample Analysis:

  • IHC analysis of CHCHD6 expression in tumor biopsies (recommended dilution: 1:20-1:200)

  • Correlation with clinical outcomes and treatment response

  • Evaluation of CHCHD6 as a potential prognostic marker

These approaches can help establish whether CHCHD6 modulation represents a viable strategy for enhancing tumor sensitivity to established chemotherapeutics, potentially enabling lower dosing and reduced side effects.

How can I combine CHCHD6 antibodies with metabolic analysis to understand mitochondrial bioenergetics?

Integrating CHCHD6 protein analysis with functional metabolic assays provides comprehensive insights:

• Respiratory Chain Analysis:

  • Oxygen consumption rate (OCR) measurements in cells with altered CHCHD6 levels

  • CHCHD6 knockdown causes reductions in oxygen consumption

  • Correlation between CHCHD6 protein levels (by Western blot) and respiratory capacity

• ATP Production Assessment:

  • Quantification of cellular ATP in CHCHD6 modulated systems

  • CHCHD6 knockdown leads to decreased intracellular ATP content

  • Real-time ATP monitoring using luciferase-based reporters

• Mitochondrial Membrane Potential Analysis:

  • Use of potentiometric dyes combined with flow cytometry or microscopy

  • CHCHD6-knockdown cells show decreased mitochondrial membrane potential (ΔΨm)

  • Correlation between CHCHD6 protein levels and membrane potential maintenance

• Metabolomic Profiling:

  • Global metabolite analysis in CHCHD6 knockout vs. control cells

  • Focus on TCA cycle intermediates and related pathways

  • Integration with proteomics data to identify compensatory mechanisms

These combined approaches have revealed that while CHCHD6 knockout affects cristae density and organization, the functional impact on bioenergetics is less severe than with Mitofilin knockdown, suggesting partial functional redundancy within the MICOS complex .