CHCHD3 Antibody

What is CHCHD3 and what is its biological significance?

CHCHD3, also known as MIC19, is an inner mitochondrial membrane scaffold protein that functions as part of the mitochondrial contact site and cristae organizing system (MICOS). This 26.152 kDa protein plays a critical role in maintaining the structural integrity of mitochondrial cristae. The absence of CHCHD3 leads to significant reductions in ATP production, cell growth, and oxygen consumption . CHCHD3 contains coiled-coil-helix-coiled-coil-helix domains and has several alternative names including FLJ20420, MICOS19, MINOS3, Mic19, and PPP1R22 . The protein has 227 amino acids with a calculated molecular weight of 26 kDa, which corresponds closely to the observed molecular weight in experimental systems .

What applications are validated for CHCHD3 antibodies?

CHCHD3 antibodies have been extensively validated for multiple research applications:

The antibodies have demonstrated consistent performance across these applications, with particular strength in Western blot analyses where they detect a specific band at approximately 26 kDa .

What species reactivity do CHCHD3 antibodies demonstrate?

CHCHD3 antibodies have been tested and validated for reactivity with human, mouse, and rat samples . Positive Western blot detection has been demonstrated in multiple cell lines and tissue types including:

• Human: HEK-293 cells, placenta tissue, A431 cells, HeLa cells, Jurkat cells, liver cancer tissue

• Mouse: Liver tissue, NIH/3T3 cells, heart tissue

• Rat: Liver tissue, RH-35 cells, heart tissue

This cross-species reactivity makes these antibodies versatile tools for comparative studies across mammalian models.

What are the recommended dilutions for different applications of CHCHD3 antibody?

Optimized dilution recommendations vary by application technique:

It is strongly recommended that researchers titrate these antibodies in each specific testing system to obtain optimal results, as the actual working concentration can vary based on sample type and experimental conditions .

What antigen retrieval methods are optimal for CHCHD3 IHC staining?

For immunohistochemical applications with CHCHD3 antibodies, heat-mediated antigen retrieval methods have been validated. Two buffer systems have demonstrated effectiveness:

• Primary recommended method: TE buffer at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

• EDTA buffer at pH 8.0 (epitope retrieval solution)

Validation studies using paraffin-embedded sections of human liver cancer tissue, rat heart tissue, and mouse heart tissue demonstrate successful staining following heat-mediated antigen retrieval in EDTA buffer (pH 8.0) . The protocol typically involves blocking with 10% goat serum, overnight antibody incubation at 4°C, and detection using peroxidase-conjugated secondary antibodies with DAB as the chromogen .

What are the optimal storage conditions for CHCHD3 antibodies?

For long-term stability and activity maintenance:

• Long-term storage: Store at -20°C, where the antibody remains stable for up to one year

• Working storage: For frequent use over shorter periods (up to one month), storage at 4°C is acceptable

• Formulation: The antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting: For the -20°C storage concentration provided (typically 1mg/ml), aliquoting is generally unnecessary

• Avoid repeated freeze-thaw cycles to maintain antibody integrity and performance

Some formulations contain 0.1% BSA in smaller size offerings (20μl), which should be noted when planning experiments .

How can CHCHD3 antibodies be used for sub-mitochondrial localization studies?

CHCHD3/MIC19 antibodies can be effectively employed in sophisticated sub-mitochondrial localization studies. A methodologically rigorous approach involves:

• Genetic tagging of CHCHD3/MIC19 with reporter proteins such as miniSOG or APEX2 for high-resolution localization

• Using antibodies against CHCHD3 in conjunction with these tagged constructs for validation

• Co-immunoprecipitation studies to identify interaction partners within the MICOS complex

• Immunofluorescence combined with super-resolution microscopy techniques

Research has demonstrated successful genetic tagging approaches using constructs like Mic19-105–miniSOG and Mic19-133–miniSOG to precisely locate this protein within mitochondrial subcompartments . For these advanced applications, co-staining with other mitochondrial markers like Mic60 (Mitofilin) and Sam50 provides contextual information about CHCHD3's role in the MICOS complex architecture .

How can I design experiments to study CHCHD3's role in the MICOS complex?

For investigating CHCHD3's functional role in MICOS complex assembly and function:

• Co-immunoprecipitation experiments using CHCHD3 antibodies can pull down interacting MICOS components:

  • Use 1:200 dilution of antibody for immunoprecipitation experiments

  • Include appropriate controls such as IgG isotype controls

  • Analyze by Western blot with antibodies against other MICOS components (e.g., Mic60/Mitofilin)

• Knockout/knockdown approaches followed by rescue experiments:

  • Analyze changes in mitochondrial morphology and function

  • Use CHCHD3 antibodies to confirm knockdown/knockout efficiency

  • Implement tagged CHCHD3 constructs for rescue experiments and track using both tag-specific and CHCHD3-specific antibodies

• Subcellular fractionation approaches to monitor MICOS complex integrity:

  • Use differential centrifugation to isolate mitochondria

  • Further fractionate into membrane and soluble components

  • Analyze distribution of CHCHD3 and interacting partners by Western blot

These experimental approaches can help elucidate CHCHD3's role in maintaining cristae morphology and mitochondrial function .

What are the most effective validation approaches for CHCHD3 antibody specificity?

Rigorous validation of CHCHD3 antibody specificity is critical for accurate experimental interpretation:

• Genetic approaches:

  • CHCHD3 knockdown/knockout using siRNA or CRISPR-Cas9

  • Overexpression of tagged CHCHD3 constructs

  • Comparison of staining/blotting patterns between modified and control cells

• Peptide competition assays:

  • Pre-incubation of antibody with immunizing peptide (e.g., CHCHD3 fusion protein Ag22579)

  • Parallel staining/blotting with blocked and unblocked antibody

  • Reduction or elimination of specific signal confirms specificity

• Cross-validation with multiple antibodies:

  • Compare results from antibodies raised against different CHCHD3 epitopes

  • Use both monoclonal and polyclonal antibodies when available

  • Confirm consistency of localization and molecular weight detection

• Mass spectrometry validation:

  • Immunoprecipitate CHCHD3 from cellular lysates

  • Confirm identity of pulled-down proteins by mass spectrometry

  • Verify presence of known CHCHD3 peptides and interacting partners

Why might I observe variable band patterns in Western blots using CHCHD3 antibody?

When Western blot results with CHCHD3 antibody show unexpected patterns, consider these potential explanations:

• Post-translational modifications:

  • CHCHD3 may undergo modifications affecting migration patterns

  • Different cell/tissue types may exhibit different modification profiles

  • Consider phosphatase treatment to eliminate phosphorylation-based mobility shifts

• Alternative splicing:

  • Several transcript variants encoding different isoforms exist for CHCHD3

  • Tissue-specific expression of isoforms may result in different banding patterns

  • Consult transcript databases to identify expected sizes of alternative isoforms

• Sample preparation effects:

  • Proteolytic degradation may generate fragments

  • Heating conditions can affect migration of mitochondrial membrane proteins

  • Modify lysis buffer composition to include appropriate protease inhibitors

• Non-specific binding:

  • Optimize blocking conditions (5% non-fat milk/TBS is standard)

  • Adjust antibody concentration based on signal-to-noise ratio

  • Increase washing duration/frequency with TBS-0.1% Tween

Reference Western blots show a clear band at approximately 26 kDa in multiple cell lines (HEK-293, HeLa, Jurkat, NIH/3T3) and tissues (human placenta, mouse liver) .

What controls should be included in CHCHD3 antibody experiments?

Robust experimental design requires appropriate controls:

• Technical controls:

  • Isotype control antibody (rabbit IgG at matching concentration)

  • Secondary antibody-only control for immunofluorescence/IHC

  • Ladder/molecular weight markers spanning 15-35 kDa range for Western blot

• Biological controls:

  • Known positive samples (e.g., HeLa cells, human liver tissue)

  • CHCHD3 knockdown/knockout samples if available

  • Overexpression system with tagged CHCHD3 constructs

• Localization controls:

  • Co-staining with established mitochondrial markers (e.g., CoxIV)

  • Other MICOS components (Mic60/Mitofilin, Sam50) for co-localization studies

  • Z-stack imaging to confirm subcellular localization patterns

• Western blot optimization controls:

  • Loading control (cytoskeletal or housekeeping protein)

  • Mitochondrial marker (e.g., CoxIV) to confirm fractionation quality

  • Gradient gel analysis to improve resolution around 26 kDa

How can I optimize CHCHD3 detection in challenging tissue samples?

For tissues with high autofluorescence, high background, or weak CHCHD3 expression:

• Antigen retrieval optimization:

  • Compare TE buffer (pH 9.0) versus citrate buffer (pH 6.0) and EDTA buffer (pH 8.0)

  • Adjust retrieval times and temperatures systematically

  • Consider pressure cooker-based retrieval for particularly challenging samples

• Signal amplification strategies:

  • Implement tyramide signal amplification for immunofluorescence

  • Use biotin-streptavidin systems for IHC

  • Consider polymer-based detection systems for enhanced sensitivity

• Background reduction approaches:

  • Extend blocking time (10% goat serum standard)

  • Include carrier proteins or mild detergents in antibody diluent

  • Pre-adsorb secondary antibodies against tissue homogenates

• Technical adjustments:

  • Modify fixation protocols (duration, fixative composition)

  • Optimize section thickness for better antibody penetration

  • Implement automated staining platforms for consistency

Successful IHC has been demonstrated in human liver cancer tissue, human heart tissue, rat heart tissue, and mouse heart tissue using heat-mediated antigen retrieval followed by DAB detection .

How do different CHCHD3 antibody clones compare in performance and specificity?

When selecting between different CHCHD3 antibody options:

• Immunogen considerations:

  • Some antibodies are raised against full-length recombinant CHCHD3 (position M1-L223)

  • Others use specific fusion proteins (e.g., Ag22579)

  • The immunogen influences epitope availability in different applications

• Validation extent:

  • Consider the breadth of published applications (some antibodies have 30+ Western blot citations)

  • Review validation images provided by manufacturers

  • Evaluate the diversity of validated cell/tissue types

• Clonality considerations:

  • Polyclonal antibodies provide broader epitope recognition

  • Current commercial CHCHD3 antibodies are predominantly rabbit polyclonal

  • This format offers advantages for applications like immunoprecipitation

• Cross-reactivity profile:

  • Most validated antibodies react with human, mouse, and rat CHCHD3

  • Consider specific experimental needs for other species

  • Review aligned sequences to predict potential cross-reactivity

How can CHCHD3 antibodies be used in multi-parametric studies of mitochondrial dynamics?

For complex studies of mitochondrial structure and function:

• Multi-labeling strategies:

  • Combine CHCHD3 antibody with other MICOS component antibodies

  • Use fluorescently-tagged antibodies compatible with flow cytometry

  • Implement multiplexed approaches with markers of mitochondrial function

• Live-cell imaging approaches:

  • Correlate fixed-cell antibody staining with live-cell dynamics

  • Use split-GFP complementation with CHCHD3 constructs

  • Validate patterns observed in live cells with antibody staining

• Super-resolution microscopy applications:

  • CHCHD3 antibodies can be used with STORM, STED, or SIM microscopy

  • Focus on cristae junctions where MICOS complex localizes

  • Compare subcellular localization under different experimental conditions

• Integration with functional assays:

  • Correlate CHCHD3 distribution with measurements of:

    • Mitochondrial membrane potential

    • Respiration rate

    • ATP production

    • Cristae morphology by electron microscopy

These integrated approaches provide mechanistic insights into how CHCHD3 distribution relates to mitochondrial function .