CHCHD7 Antibody

What is CHCHD7 and why is it important in research?

CHCHD7 (Coiled-coil-helix-coiled-coil-helix domain containing 7) is a member of a multifamily of proteins containing a conserved (coiled coil 1)-(helix 1)-(coiled coil 2)-(helix 2) domain. The protein is ubiquitously expressed in human tissues, as demonstrated by Northern blot analysis . While its precise biological function remains largely unknown, CHCHD7 has gained research significance due to its involvement in chromosomal aberrations found in salivary gland pleiomorphic adenomas, the most common benign epithelial tumors of the salivary gland . CHCHD7 and PLAG1 are located head-to-head approximately 500 bp apart in chromosome 8q12, and CHCHD7-PLAG1 gene fusions have been identified in these tumors . The protein is also associated with mitochondrial function, specifically the intermembrane space , suggesting potential roles in cellular energetics.

What are the available types of CHCHD7 antibodies and their source organisms?

Currently, several types of CHCHD7 antibodies are available for research, primarily polyclonal antibodies produced in rabbits. These include:

Most commercially available antibodies are affinity-purified and supplied in buffered solutions containing glycerol and preservatives like sodium azide . These antibodies are typically raised against synthetic peptides corresponding to regions of the human CHCHD7 protein, often near the C-terminus .

What are the standard applications for CHCHD7 antibodies in research?

CHCHD7 antibodies have been validated for several standard research applications with specific recommended dilutions:

When performing these applications, researchers should note that positive WB detection has been reported in mouse pancreas tissue and human placenta tissue . For IHC, human pancreas tissue has shown positive results, with vendors suggesting antigen retrieval using TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 .

How should I optimize antigen retrieval for CHCHD7 detection in FFPE tissues?

Antigen retrieval optimization for CHCHD7 detection in formalin-fixed paraffin-embedded (FFPE) tissues requires careful consideration of buffer systems and retrieval conditions. According to available data, the primary recommended method is:

• TE buffer at pH 9.0 as the first choice for heat-induced epitope retrieval (HIER)

• Alternative option: Citrate buffer at pH 6.0

When optimizing retrieval conditions, consider implementing a systematic approach:

• Perform a temperature gradient experiment (95-125°C) with both buffer systems

• Test variable retrieval durations (10-30 minutes)

• Include positive control tissues (human pancreatic tissue is recommended based on validation data)

• After retrieval, ensure adequate cooling before proceeding with blocking steps

For challenging samples, consider dual antigen retrieval approaches where enzymatic treatment (proteinase K, 5-10 μg/mL for 5-10 minutes) follows the heat-induced method. When evaluating results, assess not only staining intensity but also the expected subcellular localization pattern in mitochondria .

What are the optimal conditions for detecting CHCHD7 by Western blot in different tissue types?

Optimizing Western blot detection of CHCHD7 requires consideration of tissue-specific parameters and protein characteristics:

Extraction Protocol:
For mitochondrial proteins like CHCHD7, use a mitochondrial isolation buffer containing:

• 250 mM sucrose

• 10 mM Tris-HCl (pH 7.4)

• 0.1 mM EGTA

• Protease inhibitor cocktail

Sample Preparation Parameters:

• Expected molecular weight: 10-12 kDa observed (calculated 12 kDa)

• Recommended loading amount: 10-30 μg of total protein

• Reducing conditions required

• Heat samples at 70°C rather than 95°C to prevent aggregation of mitochondrial proteins

Electrophoresis and Transfer:

• Use higher percentage gels (15-20%) for optimal resolution of low molecular weight CHCHD7

• Transfer to PVDF membranes (0.2 μm pore size) at lower voltage (30V) overnight at 4°C

• Add 10% methanol to transfer buffer to enhance transfer of small proteins

Detection Parameters:

• Primary antibody dilution: 1:500-1:2000

• Incubation: Overnight at 4°C

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

• Extended blocking (2 hours) with 5% non-fat milk to reduce background

• Recommended positive control: rat liver tissue lysate

Tissue-Specific Considerations:
Mouse pancreas and human placenta tissues have shown reliable detection of CHCHD7 , so consider using these as positive controls when establishing protocols for new tissue types.

What are the key considerations for using CHCHD7 antibodies in co-localization studies with mitochondrial markers?

When designing co-localization experiments to study CHCHD7's mitochondrial localization, several technical considerations should be addressed:

Antibody Selection:

• Choose CHCHD7 antibodies validated for immunofluorescence applications with documented subcellular localization patterns

• Optimal working concentration for IF applications: 0.25-2 μg/mL

• Ensure the host species differs from your mitochondrial marker antibodies to avoid cross-reactivity

Mitochondrial Markers for Co-localization:

Fixation Optimization:

• Paraformaldehyde (4%) for 10-15 minutes preserves mitochondrial morphology

• Avoid methanol fixation which can disrupt mitochondrial structure

• For membrane proteins like CHCHD7, gentle permeabilization with 0.1% Triton X-100 for 5 minutes is recommended

Image Acquisition Parameters:

• Deconvolution microscopy or confocal microscopy with Airyscan is recommended for resolving mitochondrial substructures

• Sequential scanning to avoid bleed-through

• Z-stack imaging (0.2-0.3 μm steps) for complete mitochondrial network visualization

• Consider super-resolution techniques (STED, STORM) for studying precise submitochondrial localization

Controls and Quantification:

• Include non-mitochondrial markers (e.g., ER, Golgi) as negative controls

• Perform quantitative co-localization analysis using Pearson's or Mander's coefficients

• Validate findings with biochemical fractionation followed by Western blotting

CHCHD7's reported mitochondrial intermembrane space localization suggests that experimental design should particularly focus on markers for this specific compartment.

Why might I observe inconsistent staining patterns in CHCHD7 immunohistochemistry across different tissues?

Inconsistent staining patterns in CHCHD7 immunohistochemistry can stem from several technical and biological factors:

Technical Factors:

• Fixation Variability: Different tissues may have undergone variable fixation durations. Overfixed tissues may require extended antigen retrieval (25-30 minutes) with high-pH TE buffer .

• Antibody Concentration Optimization: Different tissues may require adjusted antibody concentrations:

  • For lymphoid tissues: 1:20-1:50 dilution recommended

  • For high-expressing tissues (pancreas): 1:50-1:100 dilution

  • For low-expressing tissues: 1:20 dilution with extended incubation (overnight at 4°C)

• Detection System Sensitivity: Consider amplification systems (TSA/CARD) for tissues with low expression levels.

Biological Factors:

• Expression Level Variation: CHCHD7 is ubiquitously expressed but at varying levels across tissues .

• Isoform Expression: Different tissues may express variant isoforms with altered epitope availability.

• Post-Translational Modifications: Tissue-specific PTMs may affect antibody recognition.

Systematic Troubleshooting Approach:

• Perform titration experiments (1:10 to 1:200) on multi-tissue arrays

• Compare multiple antigen retrieval methods side-by-side

• Include positive control tissues (pancreas) on each slide

• Test multiple antibody clones/vendors targeting different epitopes

• Consider dual IF staining with mitochondrial markers to confirm specificity

To address these issues, implement standardized tissue processing protocols across samples and consider using automated IHC platforms to minimize technical variability.

How can I address non-specific bands when using CHCHD7 antibodies in Western blotting?

Non-specific bands when detecting CHCHD7 (expected size: 10-12 kDa) can be problematic but can be addressed through systematic optimization:

Common Causes and Solutions:

• Cross-reactivity with Related Proteins:

  • CHCHD family proteins share structural similarities

  • Solution: Perform peptide competition assays using the specific immunizing peptide

  • The PEP-1529 blocking peptide is available for validating PA5-34487 antibody specificity

• Sample Preparation Issues:

  • Incomplete denaturation can cause higher MW aggregates

  • Solution: Optimize sample preparation by including 8M urea in lysis buffer for complete denaturation of mitochondrial membrane proteins

• Degradation Products:

  • Lower MW bands may represent degradation products

  • Solution: Include additional protease inhibitors (especially serine and cysteine protease inhibitors) and prepare samples fresh

Optimization Protocol:

Validation Approaches:

• Use CHCHD7 recombinant protein (ab183240) as a positive control

• Confirm specificity through siRNA/shRNA knockdown experiments

• Compare staining patterns with multiple antibodies targeting different epitopes

• Consider pre-adsorption of the antibody with the immunizing peptide

For systems showing persistent background, consider using a monoclonal antibody or implementing more stringent blocking with 5% BSA + 5% normal serum matching the host species of the secondary antibody.

What control experiments should I include to validate CHCHD7 antibody specificity in my experimental system?

Comprehensive validation of CHCHD7 antibody specificity requires a multi-faceted approach incorporating several control experiments:

Positive Controls:

• Recombinant Protein Analysis:

  • Use purified recombinant CHCHD7 protein (ab183240 or commercially available options)

  • Expected size: ~14 kDa for His-tagged recombinant protein

  • Create a standard curve with defined protein amounts (1-100 ng)

• Verified Tissue Controls:

  • Mouse pancreas tissue and human placenta tissue have confirmed expression

  • Rat liver tissue lysate is recommended as a positive control for Western blot

Negative Controls:

• Peptide Competition/Neutralization:

  • Pre-incubate antibody with 5-10× molar excess of immunizing peptide (PEP-1529 for PA5-34487)

  • Include both competed and non-competed antibody on the same blot/slide

  • Expected result: Specific signal should be eliminated or substantially reduced

• Genetic Knockdown/Knockout Validation:

  • siRNA or shRNA knockdown of CHCHD7 (validate knockdown efficiency by qRT-PCR)

  • CRISPR/Cas9-mediated knockout cell lines

  • Compare protein expression between control and KD/KO samples

Cross-Reactivity Assessment:

• Multiple Species Testing:

  • Compare detection in human, mouse, and rat samples (most antibodies show cross-reactivity)

  • Confirm that observed molecular weights are consistent with species-specific predictions

• Multi-Antibody Validation:

  • Test multiple antibodies targeting different epitopes

  • Concordant results from antibodies recognizing distinct regions strongly support specificity

Documentation Requirements:

• Maintain detailed records of lot numbers and validation experiments

• Include representative images of all controls in supplementary materials

• Document antibody concentration, incubation time/temperature, and detection methods

This comprehensive validation approach ensures that experimental observations can be confidently attributed to specific CHCHD7 detection rather than non-specific or artifactual signals.

What is known about CHCHD7's role in mitochondrial function and how can researchers study this aspect?

While detailed functional characterization of CHCHD7 remains limited, several lines of evidence point to its role in mitochondrial biology:

Current Knowledge:

• CHCHD7 localizes to the mitochondrial intermembrane space

• It belongs to the CHCHD protein family, several members of which are involved in mitochondrial respiration

• CHCHD7 has been annotated as COX23 cytochrome c oxidase assembly homolog , suggesting potential involvement in respiratory complex IV assembly or function

Experimental Approaches to Study CHCHD7's Mitochondrial Function:

• Subcellular Localization Studies:

  • Immunofluorescence with mitochondrial compartment-specific markers

  • Biochemical fractionation followed by Western blotting

  • Protease protection assays to confirm intermembrane space localization

• Functional Assessment After Genetic Manipulation:

  • Measure key mitochondrial parameters after CHCHD7 knockdown/knockout:

    • Oxygen consumption rate (Seahorse XF analyzer)

    • Mitochondrial membrane potential (TMRM, JC-1 dyes)

    • ATP production

    • Reactive oxygen species generation

    • mtDNA copy number

• Protein Interaction Studies:

  • Immunoprecipitation followed by mass spectrometry

  • Proximity labeling approaches (BioID, APEX)

  • Yeast two-hybrid screening

  • Focus on interactions with known respiratory chain components

• Rescue Experiments:

  • Complementation studies in knockout cells with wild-type vs. mutant CHCHD7

  • Cross-species rescue to assess functional conservation

Technical Considerations:

• For oxygen consumption measurements, use permeabilized cells to directly assess respiratory complex activities

• Include appropriate controls (positive: cells with known mitochondrial defects; negative: non-targeted siRNA)

• Consider tissue-specific effects, as mitochondrial function varies across tissues

Emerging Research Directions:
Investigation into whether CHCHD7 functions similarly to other CHCHD family proteins (e.g., CHCHD3, CHCHD10) which have established roles in mitochondrial cristae organization, respiratory complex assembly, and mitochondrial DNA maintenance.

How is CHCHD7 implicated in salivary gland tumors and what experimental approaches can be used to study this connection?

CHCHD7 has been specifically implicated in salivary gland pleomorphic adenomas through a novel gene fusion mechanism:

Current Understanding:

• CHCHD7-PLAG1 gene fusion is a recurrent event in pleomorphic salivary gland adenomas

• This fusion results from cryptic, intrachromosomal 8q rearrangements

• CHCHD7 and PLAG1 are located head-to-head approximately 500 bp apart in chromosome 8q12

• The breakpoints occur in the 5′-noncoding regions of the genes, leading to activation of PLAG1 by promoter swapping/substitution

• PLAG1 protein is overexpressed in epithelial, myoepithelial, and mesenchymal-like tumor cells in fusion-positive tumors

Experimental Approaches to Study the CHCHD7-PLAG1 Connection:

• Detection of CHCHD7-PLAG1 Fusion:

  • RT-PCR using primers spanning the fusion junction

  • FISH analysis using break-apart probes

  • RNA sequencing to identify fusion transcripts

  • Long-read sequencing for detailed breakpoint characterization

• Functional Studies:

  • Reporter assays to study promoter activity of CHCHD7 driving PLAG1 expression

  • CRISPR-mediated recreation of the fusion in normal salivary gland cells

  • Transcriptomic analysis comparing fusion-positive vs. fusion-negative tumors

  • Chromatin immunoprecipitation to study altered transcription factor binding

• Clinical Correlation Studies:

  • IHC analysis of PLAG1 protein expression in patient samples

  • Correlation of fusion status with clinical parameters (tumor size, recurrence, etc.)

  • Comparison with other known genetic alterations in pleomorphic adenomas

Methodological Considerations:

• Due to the cryptic nature of these rearrangements, conventional cytogenetics may miss the CHCHD7-PLAG1 fusion

• Combined approaches (FISH on interphase nuclei and nuclear chromatin fibers) may be necessary for detection

• Western blot and IHC analyses should be used to confirm PLAG1 protein overexpression

Research Implications:
The identification of CHCHD7-PLAG1 fusion emphasizes the significance of PLAG1 activation in pleomorphic adenomas and demonstrates that the gene is more frequently activated than previously anticipated . Future studies could explore whether targeting this fusion or its downstream effects might have therapeutic potential.

How can researchers explore the potential functional differences between CHCHD7 and other CHCHD family proteins?

The CHCHD protein family shares a conserved (coiled coil 1)-(helix 1)-(coiled coil 2)-(helix 2) domain structure, but members appear to have diverse functions. Exploring the functional distinctions between CHCHD7 and other family members requires a systematic comparative approach:

Sequence and Structure Analysis:

• Comparative Sequence Analysis:

  • Multiple sequence alignment of CHCHD family proteins

  • Identification of conserved vs. variable regions

  • Evolutionary analysis to determine functional divergence points

• Structural Modeling:

  • Homology-based structural prediction for CHCHD7

  • Comparison with known structures of other CHCHD proteins

  • Identification of potential functional motifs unique to CHCHD7

Experimental Approaches:

• Localization Studies:

  • Comparative subcellular localization of all CHCHD family proteins using consistent methodology

  • Super-resolution microscopy to determine precise submitochondrial localization

  • Create chimeric proteins swapping domains between CHCHD7 and other family members to identify localization determinants

• Interactome Mapping:

  • Systematic immunoprecipitation of each CHCHD protein followed by mass spectrometry

  • Proximity labeling (BioID, APEX) of each family member

  • Network analysis to identify shared vs. unique interaction partners

  • Focus on mitochondrial function-related interactors

• Functional Redundancy Assessment:

  • Single and combinatorial knockdown/knockout of CHCHD family members

  • Cross-complementation experiments (can one family member rescue defects caused by loss of another?)

  • Phenotypic profiling (growth, mitochondrial function, stress responses)

• Expression Pattern Analysis:

  • Comprehensive tissue expression profiling of all family members

  • Response to cellular stressors (oxidative stress, hypoxia, nutrient deprivation)

  • Developmental expression patterns

Technical Considerations:

• Use epitope-tagged versions of each protein where antibody cross-reactivity is a concern

• Include proper controls for overexpression artifacts

• Consider tissue-specific functions in experimental design

Emerging Research Questions:

• Does CHCHD7 participate in the same mitochondrial processes as other family members or has it evolved distinct functions?

• Are there functional redundancies that explain why single gene mutations/knockouts may have subtle phenotypes?

• How do post-translational modifications differ between family members and affect function?

Investigating these questions will provide insights into the specific role of CHCHD7 in the context of this protein family and broader mitochondrial biology.

What are the key epitope considerations when selecting a CHCHD7 antibody for specific research applications?

Selecting the optimal CHCHD7 antibody requires careful consideration of epitope characteristics based on the intended application:

Epitope Mapping of Available Antibodies:

Application-Specific Epitope Considerations:

• Western Blotting:

  • Linear epitopes are preferred (all available antibodies suitable)

  • Consider epitope accessibility in denatured conditions

  • Avoid antibodies targeting regions prone to post-translational modifications

  • C-terminal antibodies may be preferred for detecting full-length protein vs. truncated forms

• Immunohistochemistry/Immunofluorescence:

  • Epitope must remain accessible after fixation and antigen retrieval

  • Antibodies recognizing native conformational epitopes may provide better results

  • Consider epitope conservation if detecting CHCHD7 across species

  • HPA050783 has been validated for IHC/IF applications

• Immunoprecipitation:

  • Epitope must be accessible in the protein's native conformation

  • Avoid epitopes that might be masked by protein-protein interactions

  • Consider using antibodies recognizing different epitopes for IP and detection

Peptide Design for Custom Antibodies:
When designing custom antibodies against CHCHD7, consider:

• Regions with high antigenicity and surface probability

• Avoidance of hydrophobic regions (amino acids 70-85)

• Exclusion of regions with high sequence similarity to other CHCHD family members

• Inclusion of the CHCH domain for functional studies, but with careful validation against other family members

Cross-Reactivity Considerations:

• CHCHD7 shares structural similarities with other CHCHD family proteins

• When studying novel tissues/species, validate antibody specificity using recombinant protein controls

• Consider epitope conservation across species for comparative studies

Proper epitope selection based on the intended application will significantly enhance experimental outcomes and data reliability.

How do different validation methods compare when assessing CHCHD7 antibody specificity?

Different validation methods provide complementary evidence for antibody specificity, each with distinct advantages and limitations. A comprehensive validation approach for CHCHD7 antibodies should incorporate multiple methods:

Comparison of Validation Approaches:

Integrated Validation Strategy for CHCHD7 Antibodies:

• Primary Validation (Minimum Requirements):

  • Western blot showing single band at expected MW (10-12 kDa)

  • Peptide competition showing signal elimination

  • Positive control (rat liver tissue lysate)

• Secondary Validation (Enhanced Confidence):

  • siRNA knockdown with quantified reduction in signal

  • Recombinant protein detection

  • Concordant results with ≥2 antibodies

• Tertiary Validation (Highest Confidence):

  • Knockout cell line/tissue

  • Mass spectrometry confirmation of immunoprecipitated protein

  • Cross-species conservation of detection pattern

Quantitative Assessment:
Document validation results with quantitative metrics:

• Signal-to-noise ratio

• Percent signal reduction in competition/knockdown

• Correlation coefficients between multiple antibodies

• Detection sensitivity (minimum protein amount detectable)

Implementing this multi-level validation strategy provides robust evidence for CHCHD7 antibody specificity, enhancing experimental reproducibility and data interpretation confidence.