CHCHD2 Antibody

What is CHCHD2 and why is it important in research?

CHCHD2 (Coiled-coil-helix-coiled-coil-helix domain containing 2) is a mitochondrial protein that plays crucial roles in:

• Regulating mitochondrial respiration through complex IV stability

• Maintaining mitochondrial cristae structure

• Controlling apoptotic pathways through interaction with BCL-xL

• Functioning in complex with CHCHD10

Its significance has dramatically increased since mutations in CHCHD2 were linked to autosomal dominant Parkinson's disease (PD) and potentially Alzheimer's disease/frontotemporal dementia. CHCHD2 shares 58% amino acid sequence identity with CHCHD10, which is associated with amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) when mutated .

What is the subcellular localization pattern of CHCHD2?

CHCHD2 demonstrates dual localization patterns:

• Primary localization in mitochondria (intermembrane space)

• Secondary localization in the nucleus under certain conditions

When using immunofluorescence techniques, endogenous CHCHD2 appears in both mitochondria and nucleus, while overexpressed non-tagged CHCHD2 shows a similar pattern with less nuclear distribution. The protein shows little overlap with endoplasmic reticulum (ER, detected by KDEL antibody) or with the autophagy-lysosomal system (detected by LAMP1 antibody) . For optimal visualization of mitochondrial localization, super-resolution microscopy has been particularly valuable in identifying CHCHD2's proximity to MICOS (mitochondrial contact site and cristae organizing system) .

What applications are CHCHD2 antibodies validated for?

Current commercial CHCHD2 antibodies are validated for multiple applications:

These applications have been validated across human and mouse samples, with observed molecular weight typically around 16-18 kDa .

How should CHCHD2 and CHCHD10 interaction be studied experimentally?

To investigate CHCHD2-CHCHD10 interactions, researchers should consider:

Crosslinking approaches:

• Use disuccinimidyl glutarate (DSG) or disuccinimidyl suberate (DSS) crosslinkers

• Western blot analysis reveals two CHCHD2 immunoreactive bands near 37 kDa:

  • A CHCHD2 homodimer

  • A more abundant heterodimer composed of CHCHD2 and CHCHD10

Co-immunoprecipitation strategy:

• Express tagged versions (e.g., CHCHD2-Flag) in stable cell lines

• CHCHD2-Flag efficiently pulls down both endogenous CHCHD2 and CHCHD10

• CHCHD10-Flag pulls down endogenous CHCHD2 but less endogenous CHCHD10 compared to CHCHD2-Flag

This interaction pattern indicates specific complex formation between these proteins and their potential functional redundancy .

What controls are essential when validating CHCHD2 antibody specificity?

Rigorous validation requires multiple controls:

• CHCHD2 knockout cells/tissues:

  • The most definitive control showing absence of signal in WB, IF, and IHC

  • CRISPR-Cas9 generated CHCHD2 KO cell lines are ideal reference standards

• Peptide competition assays:

  • Pre-incubate antibody with immunogenic peptide to block specific binding

• Multiple antibody comparison:

  • Test antibodies raised against different epitopes (N-terminal vs. C-terminal)

  • Consistent results across antibodies confirm specificity

• Positive controls:

  • Include known CHCHD2-expressing samples (HepG2, HEK-293, or mouse brain)

  • Expected molecular weight should be 16-18 kDa

• Recombinant protein standards:

  • Use purified CHCHD2 protein as size reference

Several publications have validated CHCHD2 antibodies using knockout models, confirming specificity for detection of this protein in various experimental contexts .

What are the optimal tissue preparation methods for CHCHD2 immunohistochemistry?

For optimal CHCHD2 detection in tissue sections:

Fixation and embedding:

• 4% paraformaldehyde fixation followed by paraffin embedding preserves epitope accessibility

Antigen retrieval methods:

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

Blocking conditions:

• 5% skim milk in TBS-T for 1 hour at room temperature prior to primary antibody application

Primary antibody incubation:

• Dilution range: 1:50-1:500

• Incubate overnight at 4°C with continuous shaking

• Use antibody diluent containing TBS-T buffer with 0.01% sodium azide

Detection systems:

• HRP-conjugated secondary antibodies (1:1000) with ECL detection system

• For fluorescent detection, use appropriate fluorophore-conjugated secondary antibodies

This protocol has been successfully utilized in studies of mouse brain tissue and human cancer samples .

Why might CHCHD2 antibody show inconsistent molecular weight in Western blots?

Several factors can explain molecular weight variations:

• Post-translational modifications:

  • Phosphorylation or other modifications can alter electrophoretic mobility

• Protein-protein interactions:

  • Incomplete sample denaturation can maintain stable protein complexes

  • Treatment with reducing agents like DTT and heat denaturation at 100°C for 10 minutes is recommended

• Incomplete processing:

  • As a mitochondrial protein, CHCHD2 may retain its transit peptide in some preparations

• Crosslinking effects:

  • When analyzing CHCHD2-CHCHD10 interactions using crosslinkers, CHCHD2 migrates as dimers near 37 kDa

  • These include CHCHD2 homodimers and CHCHD2-CHCHD10 heterodimers

• Splice variants:

  • Alternative splicing results in multiple transcript variants with different molecular weights

The expected molecular weight range for monomeric CHCHD2 is 16-18 kDa, while dimeric forms appear around 37 kDa after crosslinking .

How can background signal be reduced in CHCHD2 immunofluorescence experiments?

To achieve cleaner CHCHD2 immunofluorescence staining:

• Increase blocking stringency:

  • Extend blocking time to 2 hours

  • Use 3-5% BSA with 0.1-0.3% Triton X-100 in PBS

  • Add 5-10% normal serum from the species of the secondary antibody

• Optimize primary antibody conditions:

  • Titrate antibody concentration (1:200-1:800 dilution range)

  • Extend primary antibody incubation to overnight at 4°C

  • Include 0.01% sodium azide to prevent microbial growth during long incubations

• Washing optimization:

  • Increase wash duration and number of washes (5-6 times for 5 minutes each)

  • Use PBS-T (PBS with 0.1% Tween-20) for more stringent washing

• Fixation considerations:

  • For mitochondrial proteins, 4% paraformaldehyde for 15 minutes preserves structure while maintaining epitope accessibility

  • Avoid methanol fixation which can distort mitochondrial morphology

• Confocal settings:

  • Adjust laser power and gain to minimize autofluorescence

  • Use spectral unmixing for tissues with high autofluorescence

Researchers have successfully visualized CHCHD2 in both mitochondria and nucleus using these optimized conditions .

How should researchers design experiments to study CHCHD2 mutations associated with Parkinson's disease?

A comprehensive experimental approach includes:

• Generation of isogenic cell models:

  • Use CRISPR-Cas9 to introduce PD-associated mutations (T61I, R145Q, Q126X) in human embryonic stem cells (hESCs) or iPSCs

  • This maintains identical genetic background except for the CHCHD2 mutation

• Functional assays:

  • Mitochondrial respiration (oxygen consumption rate via Seahorse analysis)

  • Mitochondrial membrane potential measurements

  • ROS production

  • ATP levels

  • Apoptosis susceptibility

  • F1F0-ATPase assembly analysis

• Structural studies:

  • Super-resolution microscopy to examine cristae morphology

  • Proximity to MICOS complex components

  • Interaction with cytochrome c

• Biochemical analyses:

  • Co-immunoprecipitation to assess protein-protein interactions (e.g., with BCL-xL)

  • Blue native PAGE to examine native complex formation

  • Crosslinking studies to assess dimer formation

• In vivo models:

  • AAV-mediated expression of mutant CHCHD2 in mouse models

  • Combine with neurotoxin models (MPTP) to assess exacerbation of PD phenotypes

Studies have demonstrated that CHCHD2 mutations impair mitochondrial function and can worsen behavioral deficits and dopaminergic neurodegeneration in animal models .

What approaches are recommended to study the functional redundancy between CHCHD2 and CHCHD10?

To investigate functional overlap between these paralogous proteins:

• Genetic manipulation strategies:

  • Generate single knockouts (CHCHD2 KO, CHCHD10 KO)

  • Generate double knockouts (CHCHD2/10 DKO)

  • Perform rescue experiments with wild-type or mutant versions of either protein

• Readouts for functional assessment:

  • Monitor steady-state levels of cytochrome c oxidase (COX) subunits, particularly COX2

  • Measure complex IV assembly using blue native PAGE

  • Assess mitochondrial cristae morphology

  • Evaluate apoptotic susceptibility

• Interaction studies:

  • Use crosslinking agents (DSG or DSS) to stabilize protein complexes

  • Perform co-immunoprecipitation with tagged versions

  • Analyze migration patterns on western blots

• Localization analysis:

  • Use super-resolution microscopy to examine co-localization patterns

  • Implement proximity ligation assays to confirm in situ interactions

Research has shown that CHCHD2 and CHCHD10 exhibit functional redundancy in maintaining COX2 levels and complex IV assembly, with double knockout cells showing more severe phenotypes than single knockouts .

How can researchers effectively study CHCHD2's role in the integrated stress response (ISR)?

To investigate CHCHD2's function in the ISR pathway:

• Experimental design:

  • Generate CHCHD2 and/or CHCHD10 knockdown cells

  • Apply stress conditions (e.g., CCCP treatment for 24 hours)

  • Compare responses between control, single knockdown, and double knockdown conditions

• Molecular readouts:

  • mRNA level analysis (RT-qPCR):

    • ATF3, ATF4, DDIT3, CHAC1

  • Protein level analysis (Western blotting):

    • ASNS, PCK2, PSPH, phosphorylated eIF2α

• Stress induction methods:

  • Mitochondrial stress: CCCP (mitochondrial uncoupler)

  • ER stress: Tunicamycin or Thapsigargin

  • Oxidative stress: Hydrogen peroxide

• Time course analysis:

  • Monitor changes at multiple timepoints (6h, 12h, 24h, 48h)

  • Assess both acute and chronic responses

• Rescue experiments:

  • Re-express wild-type or mutant CHCHD2/CHCHD10 to determine which domains are critical for ISR regulation

Studies have demonstrated that CHCHD2 and CHCHD10 can regulate the integrated stress response under normal conditions and under mitochondrial stress induced by CCCP .

How does CHCHD2 function differ between cancer models and neurodegenerative disease models?

CHCHD2 exhibits context-dependent functions with important distinctions:

In cancer models:

• Acts as an anti-apoptotic protein through BCL-xL interaction

• Promotes cell proliferation and migration

• Enhances mitochondrial respiration

• Co-amplified with EGFR in non-small cell lung cancer

• Associated with increased MMP2 expression and angiogenesis

• Correlated with cancer progression in renal cell carcinoma

• Expression correlates with poor prognosis in multiple cancer types

In neurodegenerative disease models:

• Mutations (T61I, R145Q, Q126X) impair mitochondrial function

• Mutation-bearing cells show abnormal cristae morphology

• In PD models, mutations disrupt F1F0-ATPase assembly

• May not interact with BCL-xL in stem cells, contrary to cancer cells

• Loss of function leads to impaired neuroectodermal differentiation

• Contributes to mitochondrial dysfunction when mutated

This dual role makes CHCHD2 a fascinating target for both cancer and neurodegeneration research, requiring careful experimental design specific to each disease context .

What are the key methodological considerations when studying CHCHD2 in human pluripotent stem cell models?

When working with hPSCs (hESCs or iPSCs) for CHCHD2 research:

• Culture adaptation monitoring:

  • CHCHD2 expression levels serve as a marker for culture adaptation

  • Repression of CHCHD2 may indicate "survival trait acquisition" after repetitive enzymatic dissociation

  • Monitor expression alongside other adaptation markers like BCL-xL induction or CNV at 20q11.21

• Single-cell dissociation protocols:

  • CHCHD2 knockout enhances survival after single-cell dissociation

  • For consistent results, standardize dissociation methods (enzymatic vs. mechanical)

  • Include ROCK inhibitor (Y-27632) when working with wild-type cells

• Differentiation capacity assessment:

  • CHCHD2 loss impairs neuroectodermal lineage differentiation

  • Monitor PAX6 expression during spontaneous differentiation

  • Compare differentiation potential between wild-type and CHCHD2-mutant lines

• Genetic engineering considerations:

  • Use isogenic CRISPR-Cas9 edited lines to avoid confounding genomic variations

  • Implement doxycycline-inducible systems for temporal control of CHCHD2 expression

  • Validate mutant protein expression and localization using immunofluorescence

• Specialized assays:

  • Assess mitochondrial function using Seahorse XF analyzers

  • Monitor cell death susceptibility to genotoxic stressors (doxorubicin, YM155)

  • Examine ROCK activity as it may affect cell survival independent of BCL-xL interaction

Research has demonstrated that CHCHD2's role in stem cells differs from cancer cells, particularly regarding BCL-xL interaction and effects on apoptosis .

What techniques are recommended to study CHCHD2's role in mitochondrial DNA maintenance?

To investigate CHCHD2's involvement in mtDNA regulation:

• mtDNA copy number analysis:

  • Use qPCR targeting mitochondrial genes relative to nuclear genes

  • Compare control tissues with those from CHCHD2 knockout models

  • Assess multiple tissues (heart, skeletal muscle, brain) as effects may be tissue-specific

• mtDNA deletion detection:

  • Long-range PCR to identify large-scale deletions

  • Next-generation sequencing for comprehensive deletion mapping

  • Single-molecule PCR for low-frequency deletion detection

• mtDNA transcription analysis:

  • RT-qPCR measuring levels of mtDNA transcripts

  • Northern blotting for processing defects

  • RNA-seq for global mitochondrial transcriptome analysis

• In situ detection of respiratory chain deficiency:

  • Cytochrome c oxidase (COX) histochemistry

  • Combined COX/SDH histochemistry to identify COX-deficient cells

  • Quantify COX-deficient crypts in intestinal tissue sections

• Aging effects assessment:

  • Longitudinal analysis at different ages (3, 6, 12 months)

  • Compare mtDNA maintenance parameters between young and aged animals

  • Correlate with phenotypic changes

Current research indicates that 12-month-old Chchd2-deficient mice accumulate COX-deficient colonic crypts, but show no changes in mtDNA copy number or mtDNA transcript levels in heart and skeletal muscle .

How can CHCHD2 antibodies be utilized in biomarker development for Parkinson's disease?

For developing CHCHD2-based biomarkers in PD:

• Tissue-based approaches:

  • Immunohistochemical analysis of post-mortem brain sections

  • Evaluation of CHCHD2 expression patterns in PD vs. control brains

  • Assessment of CHCHD2 aggregation or mislocalization in disease states

• Fluid biomarker development:

  • Detection of CHCHD2 levels in cerebrospinal fluid

  • Analysis of CHCHD2 in blood plasma/serum exosomes

  • Correlation with disease progression and severity

• Multi-marker panels:

  • Combine CHCHD2 with other mitochondrial markers (CHCHD10, PINK1, Parkin)

  • Integrate with established PD biomarkers (α-synuclein, DJ-1)

  • Develop algorithms incorporating multiple protein measurements

• Validation considerations:

  • Include antibodies targeting different CHCHD2 epitopes

  • Ensure consistent performance across patient cohorts

  • Establish sensitivity/specificity in distinguishing PD from other neurodegenerative conditions

• Technical considerations:

  • Standardize sample collection and processing protocols

  • Implement highly sensitive detection methods (ELISA, digital ELISA, mass spectrometry)

  • Control for confounding factors (age, comorbidities, medications)

The association of CHCHD2 mutations with autosomal dominant PD makes this protein a promising candidate for biomarker development, though further validation studies are needed to establish clinical utility .

What experimental approaches can identify potential therapeutic targets in the CHCHD2 pathway for neurodegenerative diseases?

To discover therapeutic interventions targeting CHCHD2:

• Drug screening platforms:

  • High-throughput screens using CHCHD2 mutant cell lines

  • Readouts: mitochondrial function, cell viability, protein aggregation

  • Repurposing FDA-approved compounds with mitochondrial targets

• Therapeutic candidate examples:

  • Elamipretide (SS-31): a mitochondria-targeted peptide that may benefit patients with CHCHD2 mutations

  • Compounds enhancing mitochondrial biogenesis (e.g., AICAR, resveratrol)

  • Modulators of the mitochondrial unfolded protein response

• Target validation approaches:

  • Genetic rescue experiments in disease models

  • Restoration of CHCHD2-CHCHD10 complex formation

  • Stabilization of mitochondrial cristae structure

  • Enhancement of respiratory chain complex assembly

• Translational considerations:

  • Assess blood-brain barrier penetration

  • Evaluate safety in long-term administration

  • Develop biomarkers for treatment response

  • Design combinatorial approaches targeting multiple pathways

• Model systems for evaluation:

  • Patient-derived iPSCs differentiated to neurons

  • CRISPR-engineered animal models expressing PD-linked CHCHD2 mutations

  • Combination with environmental stressors or aging to accelerate phenotypes

Research suggests the CHCHD2-CHCHD10 complex may represent a novel therapeutic target for PD and related neurodegenerative disorders, with potential benefit from mitochondria-targeted compounds like Elamipretide .

How can researchers effectively characterize CHCHD2 mutations in clinical samples?

For comprehensive analysis of CHCHD2 mutations in patient samples:

• Genetic screening methods:

  • Targeted sequencing of CHCHD2 coding regions

  • Inclusion in neurodegenerative disease gene panels

  • Whole exome/genome sequencing for novel variant discovery

• Functional characterization:

  • Fibroblast isolation from mutation carriers

  • iPSC generation and neuronal differentiation

  • Assessment of mitochondrial parameters:

    • Respiration (OCR)

    • Membrane potential

    • ROS production

    • Cristae morphology

• Protein analysis in patient samples:

  • Western blotting for expression levels and post-translational modifications

  • Immunohistochemistry in biopsy or autopsy tissue

  • Analysis of protein-protein interactions (co-IP with CHCHD10, BCL-xL)

• Clinical correlation studies:

  • Genotype-phenotype correlation

  • Age of onset analysis

  • Progression rate assessment

  • Response to treatments

• Population-specific considerations:

  • Different CHCHD2 mutations have been reported in Asian versus Caucasian populations

  • Evaluation of population-specific effects on penetrance and expressivity