COX7A2L Antibody

What is COX7A2L and what is its biological function?

COX7A2L is a mitochondrial protein encoded by the COX7A2L gene. It plays a crucial role in the regulation of oxidative phosphorylation and energy metabolism. The protein is necessary for the assembly of mitochondrial respiratory supercomplexes, particularly binding to and stabilizing complex III . COX7A2L has a calculated molecular weight of approximately 13 kDa (114 amino acids) with an observed molecular weight of 14 kDa in experimental conditions . According to structural predictions, human COX7A2L contains one transmembrane domain spanning amino acids 86-107, with most of the N-terminal part exposed to the mitochondrial matrix and a short 7-amino acid C-terminal portion facing the intermembrane space .

What applications are COX7A2L antibodies validated for?

COX7A2L antibodies have been validated for multiple research applications:

When selecting an antibody for a specific application, researchers should review the validation data provided by manufacturers and consider published literature using the same antibody for similar applications .

What species reactivity do COX7A2L antibodies show?

Most commercially available COX7A2L antibodies show reactivity with:

• Human samples

• Mouse samples

• Rat samples

This cross-reactivity has been experimentally confirmed in various tissue types across these species . When working with tissues from other species, preliminary validation is recommended as reactivity may vary between antibody products and experimental conditions.

How do different mouse strains express COX7A2L variants, and how does this affect antibody selection?

Different mouse strains express distinct COX7A2L protein isoforms that may impact antibody reactivity and experimental outcomes. CD1 mice express high levels of a 113 amino acid COX7A2L protein isoform, whereas C57BL/6 mice express lower levels of a slightly shorter 111 amino acid COX7A2L protein that is less stable .

When designing experiments with mouse models:

• Consider the specific mouse strain being used

• Review antibody epitope information to ensure recognition of your strain's COX7A2L variant

• Include appropriate controls when comparing results across different mouse strains

• Be aware that protein stability differences may affect detection sensitivity

These strain differences have functional consequences, as demonstrated in studies of respiratory complex assembly, making proper antibody selection crucial for accurate data interpretation .

What are the optimal conditions for COX7A2L co-immunoprecipitation experiments?

Co-immunoprecipitation (co-IP) studies have successfully demonstrated COX7A2L's physical interactions with respiratory complexes III and IV. For optimal co-IP experiments:

• Use digitonin for mitochondrial solubilization, as it preserves complex interactions better than harsher detergents

• Consider tagged COX7A2L constructs (e.g., MYC-DDK tags) for enhanced immunoprecipitation efficiency

• Perform reverse immunoprecipitation assays using antibodies against interacting partners (e.g., CORE2 or COX1) to validate interactions

• Be aware that COX7A2L shows higher affinity for complex III than complex IV, which may affect co-IP results

• Include appropriate controls (e.g., empty vector transfections) to distinguish specific from non-specific interactions

Research has shown that immunoprecipitation with anti-CORE2 (complex III) antibodies successfully pulls down endogenous COX7A2L, while anti-COX1 (complex IV) is less efficient, reflecting the differential binding affinities of COX7A2L for these complexes .

How can I optimize antigen retrieval for COX7A2L immunohistochemistry?

Effective antigen retrieval is critical for successful COX7A2L immunohistochemistry. Based on validated protocols:

• Primary recommendation: TE buffer pH 9.0 for heat-induced epitope retrieval

• Alternative method: Citrate buffer pH 6.0, which may be necessary for certain tissue types

• Optimization variables to consider:

  • Retrieval time (typically 10-20 minutes)

  • Temperature (95-100°C)

  • Post-retrieval cooling period

  • Blocking procedures to reduce background

COX7A2L antibodies have been successfully used for IHC detection in various tissues, including human ovary tumor tissue and breast cancer tissue . The optimal dilution range for IHC applications is typically 1:20-1:200, but this should be empirically determined for each tissue type and experimental condition .

What approach should I use to validate COX7A2L antibody specificity?

Thorough validation of antibody specificity is essential for reliable COX7A2L research. A comprehensive validation approach includes:

• Genetic controls:

  • Use of knockout/knockdown systems (KO/KD), which have been employed in at least 5 publications with COX7A2L antibodies

  • Comparison between different mouse strains with known COX7A2L variants

• Technical controls:

  • Peptide competition assays using the immunizing peptide

  • Western blot analysis to confirm detection at the expected molecular weight (13-14 kDa)

  • Multiple antibody approach using antibodies targeting different epitopes

• Application-specific validation:

  • For WB: Include positive controls (e.g., mouse/rat brain tissue)

  • For IHC: Include known positive tissues and appropriate negative controls

  • For co-IP: Verify specificity through reverse IP and empty vector controls

When publishing results, clearly document the validation methods used and include RRID (Research Resource Identifier) information for the antibody (e.g., AB_2245402 for the Proteintech antibody) .

What is the role of COX7A2L in respiratory supercomplex assembly and stability?

COX7A2L plays a specific role in the assembly and stability of mitochondrial respiratory supercomplexes:

• It primarily binds to and stabilizes complex III (CIII)

• It is necessary for the formation of the III₂+IV supercomplex but does not affect respirasome formation

• Different COX7A2L protein variants affect supercomplex stability differently:

  • The 113 amino acid isoform in CD1 mice supports stable supercomplex formation

  • The 111 amino acid isoform in C57BL/6 mice results in less stable supercomplexes

• Co-immunoprecipitation studies have confirmed physical interactions with:

  • CIII subunits: CORE1, CORE2, CYC1, RISP, and UQCRQ

  • CIV subunits: COX1, COX4, COX5B, and COX6C

• Interestingly, COX7A2L levels increase in CIV-deficient conditional Lrpprc knockout mice, suggesting compensatory stabilization of CIII in response to CIV deficiency

For researchers studying mitochondrial function, these findings highlight COX7A2L as a critical factor in understanding respiratory chain organization and energy metabolism regulation.

What are the recommended storage conditions for COX7A2L antibodies?

For optimal antibody performance and longevity:

• Store at -20°C in the buffer provided by the manufacturer (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

• Most COX7A2L antibodies remain stable for one year after shipment when properly stored

• Aliquoting is generally unnecessary for -20°C storage of glycerol-containing formulations

• Some smaller size preparations (e.g., 20μl) may contain 0.1% BSA as additional stabilizer

• Avoid repeated freeze-thaw cycles that may compromise antibody activity

Always refer to the manufacturer's specific storage recommendations, as formulations may vary between suppliers.

How should I determine the optimal working dilution for my experiment?

The optimal working dilution for COX7A2L antibodies varies by application and experimental conditions:

It is strongly recommended to titrate the antibody in each testing system to obtain optimal results, as the required concentration is sample-dependent . Start with the manufacturer's recommended dilution range and perform a dilution series to determine the optimal signal-to-noise ratio for your specific experimental conditions.

What positive controls are recommended for validating COX7A2L antibody performance?

When validating COX7A2L antibodies for your experimental system, the following positive controls have been successfully used:

For Western Blot:

• Mouse brain tissue

• Rat brain tissue

For Immunohistochemistry:

• Human ovary tumor tissue

• Human breast cancer tissue

These tissues have been confirmed to express detectable levels of COX7A2L and can serve as reliable positive controls. Always include appropriate negative controls (such as isotype controls or tissues known to have low/no expression) to establish the specificity of your staining patterns.

How can I use COX7A2L antibodies to study mitochondrial dysfunction in disease models?

COX7A2L antibodies can be valuable tools for investigating mitochondrial dysfunction in various disease contexts:

• Comparative expression analysis:

  • Quantify COX7A2L levels in healthy versus diseased tissues using Western blot

  • Examine tissue distribution patterns via immunohistochemistry

  • Correlate COX7A2L expression with disease severity or progression

• Supercomplex assembly assessment:

  • Use blue native PAGE followed by Western blot to analyze supercomplex integrity

  • Combine with co-immunoprecipitation to investigate altered protein interactions

  • Compare respiratory complex stability between normal and pathological conditions

• Functional studies:

  • Monitor COX7A2L expression changes in response to metabolic stress

  • Correlate COX7A2L levels with measures of respiratory efficiency

  • Investigate compensatory responses, such as the upregulation observed in CIV-deficient models

• Genetic manipulation experiments:

  • Use knockdown/knockout approaches to model COX7A2L deficiency

  • Rescue experiments with different COX7A2L variants

  • CRISPR-Cas9 editing to introduce disease-relevant mutations

When designing these experiments, consider the differential expression of COX7A2L variants across genetic backgrounds and cell types, as these may influence experimental outcomes and interpretation .

What are the key considerations when designing co-localization studies with COX7A2L?

For successful co-localization studies involving COX7A2L:

• Antibody selection:

  • Choose antibodies raised in different host species to enable simultaneous detection

  • Verify that the COX7A2L antibody specifically labels mitochondria

  • Consider using monoclonal antibodies for improved specificity in multi-label experiments

• Mitochondrial markers:

  • Pair with established markers for specific mitochondrial compartments

  • For respiratory complex studies, combine with antibodies against CI-CV subunits

  • Include markers for both inner and outer mitochondrial membranes for precise localization

• Imaging considerations:

  • Use confocal or super-resolution microscopy for accurate co-localization assessment

  • Apply appropriate controls for bleed-through and cross-reactivity

  • Employ quantitative co-localization analysis methods (e.g., Pearson's coefficient)

• Sample preparation:

  • Optimize fixation conditions to preserve mitochondrial morphology

  • Consider mitochondrial isolation techniques for improved resolution

  • Test different permeabilization methods if access to the inner membrane is challenging

Given COX7A2L's predicted transmembrane topology (with most of the N-terminal region in the matrix and a short C-terminal portion in the intermembrane space), antibodies targeting different epitopes may show distinct localization patterns .

How does COX7A2L expression vary across different tissues and cell types?

While comprehensive tissue-specific expression data for COX7A2L was not fully detailed in the search results, we can extract key information about expression patterns:

• COX7A2L has been successfully detected in:

  • Brain tissue (mouse and rat)

  • Ovarian tumor tissue (human)

  • Breast cancer tissue (human)

• Expression levels appear to be regulated in response to mitochondrial stress conditions:

  • Increased expression has been observed in CIV-deficient conditional Lrpprc knockout mice

  • This suggests adaptive regulation of COX7A2L in response to respiratory chain deficiencies

• Expression variations exist between different mouse strains:

  • CD1 mice express higher levels of the 113 amino acid isoform

  • C57BL/6 mice express lower levels of the less stable 111 amino acid isoform

For researchers investigating tissue-specific mitochondrial function, it would be valuable to perform a systematic analysis of COX7A2L expression across different tissues and cell types, particularly in contexts of metabolic stress or disease states.

How can I use COX7A2L antibodies in studies of oxidative phosphorylation efficiency?

COX7A2L antibodies can be integrated into comprehensive studies of oxidative phosphorylation (OXPHOS) efficiency:

• Correlation studies:

  • Measure COX7A2L levels alongside oxygen consumption rates

  • Assess the relationship between COX7A2L expression and ATP production

  • Compare supercomplex stability with OXPHOS efficiency metrics

• Manipulation experiments:

  • Use RNA interference to modulate COX7A2L levels

  • Observe resulting changes in respiratory complex organization

  • Measure functional consequences on electron transfer efficiency

• Analytical approaches:

  • Combine with blue native PAGE to assess supercomplex integrity

  • Use seahorse analysis to measure respiratory parameters

  • Implement proteomics approaches to identify COX7A2L interaction partners

• Pathological contexts:

  • Examine COX7A2L's role in conditions with known OXPHOS deficiencies

  • Study compensatory mechanisms in response to complex III or IV defects

  • Investigate potential therapeutic approaches targeting supercomplex stability

Given COX7A2L's specific role in stabilizing the III₂+IV supercomplex , these studies can provide valuable insights into the relationship between supercomplex formation and respiratory efficiency in both normal physiology and disease states.