CHCHD5 Antibody

What is CHCHD5 and why is it significant in research?

CHCHD5 (coiled-coil-helix-coiled-coil-helix domain containing 5) is a mitochondrial protein with a calculated molecular weight of approximately 12 kDa (110 amino acids) . The significance of CHCHD5 lies in its emerging role in cancer research, where it has been identified as overexpressed in human malignancies, particularly breast and colon cancers . In some research contexts, CHCHD5 is also referred to as CHTM1 (a novel metabolic marker), highlighting its potential importance in cancer metabolism . The gene encoding CHCHD5 maps to human chromosome 2, which contains over 1,400 genes and constitutes about 8% of the human genome .

What types of CHCHD5 antibodies are available for research applications?

Based on current literature and commercial offerings, CHCHD5 antibodies are available in several formats:

The selection of an appropriate antibody should be based on the specific experimental requirements and the validation data available for each antibody in the intended application.

How does the structure of CHCHD5 relate to its function and antibody targeting?

CHCHD5 contains a characteristic CHCH (coiled-coil-helix-coiled-coil-helix) domain, which is a structural motif found in several mitochondrial proteins . This domain is formed by specific arrangements of cysteine residues (often in CX9C motifs) that can form disulfide bonds, contributing to protein stability and function in the mitochondrial intermembrane space . When developing or selecting antibodies, researchers should consider whether the epitope is within this domain, as it may affect antibody specificity and performance in different experimental conditions, particularly under reducing versus non-reducing conditions.

What are the recommended protocols for Western blot using CHCHD5 antibodies?

For optimal Western blot results with CHCHD5 antibodies, follow these methodological guidelines:

• Sample preparation: Prepare protein lysates from cells (A431, HeLa, HepG2, MCF-7 have shown positive detection)

• Protein loading: 20-40 μg of total protein per lane is typically sufficient

• Separation: Use 12-15% SDS-PAGE gels to efficiently resolve the low molecular weight (12 kDa) CHCHD5 protein

• Transfer: Semi-dry or wet transfer to PVDF or nitrocellulose membranes

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute CHCHD5 antibody at 1:500-1:3000 (antibody-dependent)

• Incubation: Overnight at 4°C with gentle rocking

• Washing: 3-5 times with TBST, 5-10 minutes each

• Secondary antibody: Anti-rabbit HRP-conjugated at 1:5000-1:10000

• Detection: Use ECL or similar chemiluminescent detection systems

When validating new antibodies or experimental conditions, it is highly recommended to include positive controls (known CHCHD5-expressing cells) and negative controls (CHCHD5 knockout cells) to confirm specificity.

How should immunofluorescence experiments with CHCHD5 antibodies be designed and optimized?

For successful immunofluorescence studies targeting CHCHD5:

• Cell preparation:

  • Seed cells on coverslips at 60-70% confluency

  • MCF-7 cells have been confirmed to show positive signals

• Fixation and permeabilization protocol:

  • Fix cells with 4% paraformaldehyde in PBS for 15 minutes at room temperature

  • Wash 3 times with PBS

  • Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes

• Blocking:

  • Block with PBS containing 5% BSA, 5% goat serum, and 0.01% Triton X-100 for 30 minutes

• Antibody incubation:

  • Dilute primary CHCHD5 antibody in IF buffer (PBS, 5% BSA, 0.01% Triton X-100) at 1:50-1:500

  • Incubate overnight at 4°C

  • Wash 3 times for 10 minutes each with IF buffer

  • Incubate with fluorophore-conjugated secondary antibody (typically at 1.0 μg/mL) for 1 hour at room temperature

  • Co-stain with DAPI for nuclear visualization

• Validation strategies:

  • Implement a mosaic approach mixing wildtype and knockout cells on the same coverslip to reduce staining and imaging bias

  • Include mitochondrial markers (e.g., MitoTracker or antibodies against established mitochondrial proteins) to confirm subcellular localization

What considerations are important for IHC applications of CHCHD5 antibodies?

Immunohistochemical detection of CHCHD5 requires careful attention to the following factors:

• Tissue preparation:

  • Use freshly fixed tissues or properly stored paraffin blocks

  • FFPE (formalin-fixed paraffin-embedded) sections typically 4-6 μm thick

• Antigen retrieval methods:

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

  • Optimize time and temperature for specific antibodies

• Antibody dilution and incubation:

  • Typical dilutions range from 1:20-1:100 for IHC applications

  • Overnight incubation at 4°C generally provides better results

• Controls for validation:

  • Positive controls: Tissue samples known to express CHCHD5 (e.g., thyroid or gastric tissue)

  • Negative controls: (1) Isotype-matched IgG instead of primary antibody, (2) CHCHD5-knockdown tissue or cells, (3) Antibody pre-absorbed with immunizing peptide

• Signal development and counterstaining:

  • DAB (3,3'-diaminobenzidine) detection system is commonly used

  • Hematoxylin counterstaining to visualize tissue architecture

Research has demonstrated that CHCHD5 shows increased expression in breast and colon cancer tissues compared to matched normal tissues, with approximately 81-83% of tumors exhibiting elevated levels .

How can researchers effectively validate the specificity of CHCHD5 antibodies?

A comprehensive validation strategy for CHCHD5 antibodies should include:

• Genetic validation:

  • Compare staining patterns between wildtype and CHCHD5 knockout/knockdown cells

  • The mosaic approach (mixing knockout and wildtype cells) provides direct side-by-side comparison under identical experimental conditions

• Molecular weight verification:

  • Confirm that the detected band appears at the expected molecular weight (12 kDa for human CHCHD5)

  • Be aware of potential post-translational modifications that might alter migration patterns

• Cross-reactivity assessment:

  • Test antibodies against related CHCHD family proteins (CHCHD1, CHCHD2, CHCHD3, CHCHD4, CHCHD10)

  • This is particularly important due to structural similarities within the CHCH domain family

• Peptide competition:

  • Pre-incubate antibody with the immunizing peptide/protein to block specific binding

  • This should eliminate specific signals while non-specific signals will remain

• Application-specific validation:

  • For immunoprecipitation: Verify enrichment by Western blot

  • For immunofluorescence: Confirm co-localization with established mitochondrial markers

What are the critical controls needed when studying CHCHD5 in disease models or patient samples?

When investigating CHCHD5 in disease contexts, particularly cancer, implement these essential controls:

• Paired sample controls:

  • Always use matched normal tissues/cells from the same patient when comparing disease samples

  • This controls for individual variations in baseline CHCHD5 expression

• Loading/normalization controls:

  • For Western blot: β-actin, GAPDH, or vinculin for total cell lysates

  • For mitochondrial fractions: VDAC1, Hsp60, or Tim23 are appropriate mitochondrial loading controls

• Technical replication:

  • Perform at least three independent experiments

  • For patient samples, include adequate sample sizes (n>30) for statistical power

• Methodological controls:

  • For IHC: Use isotype controls and peptide competition assays to confirm staining specificity

  • For IF: Include secondary-only controls to assess background fluorescence

• Expression validation through orthogonal methods:

  • Confirm protein expression results with mRNA expression data

  • Public databases like GEO can provide additional validation (e.g., GEO profile ID # GSD4382 for colorectal cancer CHCHD5 expression)

How should researchers approach CHCHD5 subcellular localization studies?

To accurately determine CHCHD5 subcellular localization:

• Subcellular fractionation:

  • Perform careful mitochondrial isolation using established protocols

  • Further fractionate mitochondria to separate outer membrane, inner membrane, and matrix compartments

  • Analyze CHCHD5 distribution by Western blot alongside marker proteins for each compartment

• High-resolution microscopy:

  • Confocal microscopy with co-staining for mitochondrial markers

  • Super-resolution microscopy (STED, STORM) for precise localization within mitochondrial subcompartments

• Electron microscopy:

  • Immunogold labeling for ultrastructural localization

  • This can determine if CHCHD5 associates with specific mitochondrial structures (e.g., cristae)

• Proximity labeling approaches:

  • BioID or APEX2 fusion proteins to identify proteins in close proximity to CHCHD5

  • This can provide insights into functional protein complexes

Given that CHCHD family proteins typically localize to the mitochondrial intermembrane space , particular attention should be paid to this compartment when designing localization experiments.

What are common issues in Western blot detection of CHCHD5 and how can they be resolved?

For the specific case of CHCHD5, its small size (12 kDa) requires particular attention to:

• Using higher percentage gels (15-20%)

• Optimizing transfer conditions for small proteins (lower voltage, longer time)

• Avoiding loss during membrane washing (use 0.2 μm rather than 0.45 μm pore size membranes)

How should researchers interpret conflicting results from different CHCHD5 antibodies?

When faced with contradictory results from different CHCHD5 antibodies:

• Epitope comparison:

  • Determine the epitope regions recognized by each antibody

  • Antibodies targeting different regions may give different results if:

    • Post-translational modifications mask certain epitopes

    • Protein interactions shield specific regions

    • Conformational changes affect epitope accessibility

• Validation status assessment:

  • Prioritize results from antibodies validated using knockout controls

  • Consider the extent of validation for each specific application

• Cross-validation approach:

  • Use orthogonal methods (qPCR, mass spectrometry) to resolve discrepancies

  • Employ multiple antibodies in the same experiment to identify consistent patterns

• Experimental conditions review:

  • Different fixation methods in IF/IHC may affect epitope availability

  • Denaturing vs. native conditions in Western blot and IP may explain differences

• Reporting recommendations:

  • Clearly document all antibodies used (catalog number, lot, dilution)

  • Acknowledge limitations and discrepancies in publications

  • Consider reporting results from multiple antibodies where appropriate

What factors affect CHCHD5 detection in different cell types and tissue samples?

Several factors may influence the successful detection of CHCHD5 across different biological samples:

• Expression level variations:

  • CHCHD5 shows variable expression across tissues and cell types

  • Cancer tissues generally show higher expression (81-83% of breast and colon tumors)

  • Cell lines with confirmed CHCHD5 expression include A431, HeLa, HepG2, and MCF-7

• Sample preparation impact:

  • Fixation methods and duration affect epitope preservation

  • For FFPE tissues, older blocks may require adjusted antigen retrieval protocols

  • Fresh frozen samples may provide better epitope preservation

• Mitochondrial content differences:

  • Cells with higher mitochondrial content (e.g., cardiomyocytes, hepatocytes) may show stronger signals

  • Consider normalizing to mitochondrial markers when comparing across cell types

• Disease state influences:

  • Changes in mitochondrial morphology or content in disease states may affect detection

  • Post-translational modifications may be altered in pathological conditions

• Technical considerations:

  • Different cell types may require adjusted permeabilization conditions

  • Tissue-specific autofluorescence or endogenous peroxidase activity may necessitate additional blocking steps for IF or IHC

How can CHCHD5 antibodies be employed in studying mitochondrial dysfunction in disease?

CHCHD5 antibodies can be valuable tools for investigating mitochondrial abnormalities in various diseases:

• Quantitative assessment of altered expression:

  • Western blot and image analysis to quantify CHCHD5 levels in disease vs. control samples

  • IHC to visualize expression changes in specific tissue regions or cell types

• Co-localization studies:

  • Dual immunofluorescence with markers of mitochondrial stress or damage

  • Changes in CHCHD5 localization pattern may indicate mitochondrial dysfunction

• Proximity labeling applications:

  • CHCHD5 antibodies for immunoprecipitation followed by mass spectrometry

  • Identifying altered CHCHD5 protein interactions in disease states

• Dynamic regulation analysis:

  • Antibodies to track CHCHD5 levels during disease progression or treatment response

  • Phospho-specific antibodies (if available) to detect post-translational modifications

• Therapeutic target assessment:

  • Using antibodies to monitor CHCHD5 modulation in response to experimental therapeutics

  • High-content screening with CHCHD5 immunostaining as a readout for compound effects

Given the observed overexpression of CHCHD5 in cancer tissues, particularly breast and colon cancers , these approaches are especially relevant for cancer research.

What are the methodological approaches for studying interactions between CHCHD5 and other mitochondrial proteins?

To investigate CHCHD5 protein interactions:

• Co-immunoprecipitation strategies:

  • Use CHCHD5 antibodies for pull-down experiments followed by Western blot or mass spectrometry

  • Proper controls include IgG control and CHCHD5-knockout cell extracts

  • Consider both native and crosslinked conditions to capture transient interactions

• Proximity-based approaches:

  • BioID or APEX2 fusion proteins to identify proteins in the vicinity of CHCHD5

  • FRET or BRET assays for monitoring direct protein interactions in living cells

• In situ visualization:

  • Proximity ligation assay (PLA) to visualize and quantify protein interactions at endogenous levels

  • Dual-color super-resolution microscopy to analyze co-localization at nanoscale resolution

• Biochemical characterization:

  • Size-exclusion chromatography to identify CHCHD5-containing protein complexes

  • Blue native PAGE to preserve native protein complexes for analysis

• Computational prediction validation:

  • Use antibodies to experimentally confirm predicted interactions from bioinformatic analyses

  • Focus on other CHCH domain-containing proteins (CHCHD1-10) as potential interactors

How can researchers effectively apply CHCHD5 antibodies in high-throughput screening or biomarker development?

For high-throughput applications and biomarker development:

• Tissue microarray (TMA) analysis:

  • Optimize CHCHD5 IHC protocols for TMA applications

  • Use automated imaging and quantification systems for consistent scoring

  • Apply validated scoring criteria (e.g., H-score, Allred score) for standardization

• Multiplex immunofluorescence approaches:

  • Combine CHCHD5 antibodies with other cancer or mitochondrial markers in multiplex panels

  • Use spectral unmixing or sequential staining approaches to overcome antibody compatibility issues

  • Apply machine learning algorithms for pattern recognition in complex datasets

• Liquid biopsy development:

  • Explore CHCHD5 detection in circulating tumor cells or extracellular vesicles

  • Develop sensitive detection methods (e.g., proximity-based assays) for low-abundance targets

• High-content screening applications:

  • Standardize CHCHD5 immunofluorescence for automated high-content systems

  • Develop multi-parametric readouts combining CHCHD5 with indicators of cell health, mitochondrial function, or cancer phenotypes

• Clinical validation strategies:

  • Design rigorous biomarker validation studies with appropriate statistical power

  • Consider clinical factors (tumor stage, grade, treatment history) in biomarker performance evaluation

  • Validate across multiple independent cohorts before clinical implementation

Research has demonstrated that CHCHD5 is overexpressed in 81.11% of colon cancer samples by Western blot and 81.81% by IHC, with similar findings in breast cancer (83.60% by IHC) , suggesting potential utility as a biomarker.

How does CHCHD5 relate to other members of the CHCHD protein family in structure and function?

Understanding CHCHD5 in the context of the broader CHCHD protein family:

• Structural comparisons:

  • CHCHD5, like other family members, contains the characteristic CHCH domain

  • Comparative structural analysis using antibodies against different family members can identify unique vs. conserved features

• Functional overlap assessment:

  • Investigate potential redundancy or complementary functions with other CHCHD proteins

  • CHCHD10 has been linked to neurodegenerative diseases (ALS and FTD) , while CHCHD5 shows cancer associations

• Co-expression and co-localization patterns:

  • Use antibodies against multiple CHCHD proteins to determine their relative expression across tissues

  • Investigate potential formation of heterocomplexes between family members

• Evolutionary conservation analysis:

  • Apply antibodies cross-reactive with orthologous proteins to study evolutionary conservation

  • Connect structural conservation to functional importance across species

• Differential regulation study:

  • Examine how expression of different CHCHD family members responds to cellular stresses

  • Identify unique vs. shared regulatory mechanisms

What technological advances are improving CHCHD5 antibody development and application?

Recent and emerging technologies enhancing antibody research:

• Recombinant antibody technologies:

  • Development of synthetic antibodies with improved specificity and reduced batch-to-batch variation

  • Single-domain antibodies (nanobodies) for applications requiring small probe size or intracellular expression

• Advanced validation approaches:

  • CRISPR-Cas9 knockout validation becoming the gold standard for antibody specificity

  • Comprehensive characterization across multiple applications (WB, IF, IHC, IP) as demonstrated in CHCHD10 studies

• Multimodal imaging innovations:

  • Combining antibody-based detection with other imaging modalities (e.g., metabolic imaging)

  • Correlative light and electron microscopy to connect molecular localization with ultrastructure

• Automation and standardization:

  • Automated protocols for consistent antibody performance across laboratories

  • Machine learning algorithms for objective interpretation of immunostaining results

• Single-cell applications:

  • Adapting antibodies for single-cell proteomics techniques

  • Spatial proteomics to map CHCHD5 distribution at subcellular resolution in tissue contexts

What is the significance of CHCHD5 post-translational modifications in research applications?

Post-translational modifications (PTMs) represent an important research frontier:

• Identification strategies:

  • Immunoprecipitation with CHCHD5 antibodies followed by PTM-specific mass spectrometry

  • Development of modification-specific antibodies (phospho-, acetyl-, ubiquitin-specific)

• Functional consequences:

  • Investigate how PTMs affect CHCHD5 localization, stability, or interactions

  • Determine if disease states show altered PTM patterns

• Regulatory mechanisms:

  • Study how cellular signaling pathways regulate CHCHD5 PTMs

  • Examine PTM changes in response to mitochondrial stress or metabolic alterations

• Technical considerations:

  • Optimize sample preparation to preserve labile PTMs

  • Validate that existing antibodies detect modified forms of CHCHD5

• Therapeutic implications:

  • Explore targeting enzymes that modify CHCHD5 as potential disease interventions

  • Develop screens to identify compounds that modulate specific CHCHD5 PTMs

Understanding CHCHD5 PTMs may be particularly relevant in cancer contexts, where altered post-translational modification is a common feature of dysregulated cellular signaling.

What are the most significant gaps in current CHCHD5 antibody research?

Despite progress in CHCHD5 research, several important gaps remain:

• Comprehensive validation across applications:

  • Many commercially available antibodies lack validation in multiple applications

  • Limited knockout-validation data compared to other protein targets

• Epitope mapping information:

  • Incomplete information about specific epitopes recognized by different antibodies

  • This limits understanding of differential performance in various applications

• Cross-reactivity profiles:

  • Insufficient data on potential cross-reactivity with other CHCHD family members

  • This complicates interpretation of results, especially in systems with multiple expressed CHCHD proteins

• Species reactivity limitations:

  • Most antibodies are validated only for human samples

  • Limited cross-reactivity data for model organisms restricts comparative studies

• Post-translational modification detection:

  • Few modification-specific antibodies available

  • Unknown effects of modifications on epitope recognition by existing antibodies

Addressing these gaps will require collaborative efforts between commercial antibody providers and academic researchers to generate and share validation data.

How can researchers contribute to improving CHCHD5 antibody resources for the scientific community?

Researchers can enhance the quality and availability of CHCHD5 research tools through:

• Rigorous validation and sharing:

  • Perform comprehensive validation using knockout controls and multiple applications

  • Publish detailed validation data including positive and negative results

  • Consider using standardized validation formats such as those proposed by the International Working Group for Antibody Validation

• Repository contributions:

  • Submit detailed protocols to repositories like protocols.io

  • Share raw validation data and images through appropriate databases

• Collaborative characterization:

  • Participate in multi-laboratory antibody testing initiatives

  • Compare performance of multiple antibodies against the same target

• Technology development:

  • Develop improved antibodies for underserved applications

  • Create new tools for studying CHCHD5 biology (e.g., nanobodies, aptamers)

• Education and standardization:

  • Promote best practices in antibody validation and use

  • Adopt standardized reporting formats for antibody experiments