COX6A1 Antibody

What is COX6A1 and what is its function in cellular metabolism?

COX6A1 is a subunit of cytochrome c oxidase (Complex IV), the final enzyme in the mitochondrial electron transport chain. It participates in the respiratory chain that contains multisubunit complexes (including Complex II, III, and IV) that cooperatively transfer electrons from NADH and succinate to molecular oxygen. This process creates an electrochemical gradient across the inner mitochondrial membrane that drives ATP synthesis . The COX6A1 protein has a calculated molecular weight of 9 kDa but is typically observed at approximately 9.5 kDa in experimental conditions . As part of Complex IV, COX6A1 contributes to the catalytic reduction of oxygen to water, utilizing electrons from cytochrome c in the intermembrane space and protons from the mitochondrial matrix .

Which COX6A1 antibodies have been validated for research applications?

Multiple COX6A1 antibodies have been validated for research use with varying degrees of documentation and application success. Based on citation frequency and validation data, the most extensively validated COX6A1 antibody is Proteintech's polyclonal antibody (11460-1-AP) with 11 research references, which has been confirmed for Western blot, ELISA, ICC, and IHC applications . Other validated options include:

When selecting an antibody, researchers should consider the specific experimental requirements and available validation data for their model system .

What species reactivity has been confirmed for COX6A1 antibodies?

COX6A1 antibodies have demonstrated cross-reactivity with samples from multiple species, primarily due to the high conservation of this protein across mammals. The Proteintech 11460-1-AP antibody has been tested and confirmed to react with human, mouse, and rat samples . Additional literature citations have reported successful use with pig samples as well . For the Abcam monoclonal antibody (ab110265), reactivity has been validated with human, rat, and mouse samples . When working with species not explicitly listed in validation data, researchers should consider sequence homology and potentially perform preliminary validation experiments to confirm cross-reactivity.

What are the recommended dilutions and protocols for using COX6A1 antibody in Western blot applications?

For Western blot applications using the Proteintech COX6A1 antibody (11460-1-AP), the recommended dilution range is 1:2000-1:12000 . The optimal dilution may vary depending on the sample type and protein expression level, so optimization is advised. The standard protocol involves:

• Protein extraction using appropriate lysis buffer (e.g., M-PER lysis buffer)

• Protein quantification (e.g., DC Protein Assay Kit)

• Loading approximately 50 μg of protein lysate on 4-15% polyacrylamide gradient gels

• Electrophoresis at 200V for approximately 30 minutes

• Transfer to nitrocellulose membranes at 100V for 1 hour

• Blocking and primary antibody incubation at the optimized dilution

• Detection with an appropriate HRP-conjugated secondary antibody

• Verification of loading using a control such as Ponceau S staining

When analyzing COX6A1 by Western blot, researchers should expect to observe a band at approximately 9.5 kDa, which corresponds to the observed molecular weight of this protein .

How should COX6A1 antibody be used for immunohistochemistry applications?

For immunohistochemistry (IHC) applications, the recommended dilution range for the Proteintech COX6A1 antibody is 1:50-1:500 . Optimal results have been reported with the following methodological considerations:

• Antigen retrieval is crucial for COX6A1 detection. The preferred method is using TE buffer at pH 9.0, although citrate buffer at pH 6.0 can serve as an alternative

• The antibody has been validated on human pancreatic cancer tissue, providing a positive control reference

• Standard IHC protocols involving deparaffinization, rehydration, antigen retrieval, endogenous peroxidase blocking, primary antibody incubation, and detection system application should be followed

• As with all antibodies, experimental optimization may be necessary for specific tissue types or fixation methods

Researchers should consider running appropriate controls, including no-primary antibody controls and known positive tissue samples, to validate staining specificity.

What considerations are important for immunofluorescence studies using COX6A1 antibody?

For immunofluorescence (IF) and immunocytochemistry (ICC) applications, COX6A1 antibody can be used at dilutions ranging from 1:50 to 1:500 . Successful IF/ICC staining has been documented in MCF-7 and SH-SY5Y cell lines, which can serve as positive controls . Key methodological considerations include:

• Cell fixation methods (4% paraformaldehyde or 80% methanol) may affect epitope accessibility

• Permeabilization with 0.1% PBS-Tween for approximately 20 minutes is typically effective

• Blocking non-specific binding with 10% normal serum in PBS containing 0.3M glycine

• Incubation with primary antibody at optimized concentration (typically 1 μg per 10^6 cells)

• Detection with fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488)

• Inclusion of isotype control antibodies and unlabeled samples as controls

For flow cytometry applications, successful staining has been demonstrated in HepG2 cells, with acquisition of >5,000 events using a 488nm laser and 525/30 bandpass filter .

How does COX6A1 antibody perform in studies of mitochondrial complex assembly and biogenesis?

COX6A1 antibody can serve as a valuable tool for investigating the assembly and biogenesis of mitochondrial Complex IV, providing insights into both normal physiological processes and pathological conditions. Research has revealed that cytochrome c oxidase assembly follows a sequential process where various nuclear-encoded subunits are incorporated into the growing complex containing mitochondrially-encoded core subunits (COX1, COX2, and COX3) .

When investigating assembly processes, researchers should consider:

• Early complex formation includes COX1 association with assembly factors COX14, COA3, and CMC1 to form the MITRAC complex

• COX subunits including COX4 and COX5A are incorporated after CMC1 release

• COX6A1 is incorporated in later stages of complex assembly

• Detection of assembly intermediates may require blue native PAGE rather than standard denaturing SDS-PAGE

• Knockdown studies of various assembly factors can help elucidate the precise timing of COX6A1 incorporation into the mature complex

Researchers studying mitochondrial biogenesis should be aware that mutations or knockdowns affecting earlier assembly components (like COX1, COX14, or COA3) will likely affect the detection of COX6A1 in fully assembled Complex IV.

What approaches can be used to validate COX6A1 antibody specificity for research applications?

Validating antibody specificity is crucial for reliable experimental results. For COX6A1 antibody, researchers should consider these validation approaches:

• Genetic knockdown/knockout validation: Using siRNA directed against COX6A1 or CRISPR/Cas9-mediated knockout followed by Western blot to confirm the absence or reduction of the detected band at 9.5 kDa

• Overexpression validation: Transfection with COX6A1 expression vectors should result in increased signal intensity

• Peptide competition assay: Pre-incubation of the antibody with the immunizing peptide should block specific staining

• Cross-reactivity assessment: Testing on samples from non-target species with low sequence homology to confirm specificity

• Multiple antibody verification: Using antibodies from different sources targeting different epitopes of COX6A1 to confirm consistent localization patterns

For knockout/knockdown approaches, lentivirus vector constructs containing short hairpin sequences that produce siRNA molecules directed against different regions (e.g., 5'UTR or 3'UTR) of COX6A1 can be employed, similar to the approach described for COX4I1 .

How can COX6A1 antibody be used to study mitochondrial dysfunction in pathological conditions?

COX6A1 antibody can be a valuable tool for investigating mitochondrial dysfunction in various diseases. Cytochrome c oxidase dysfunction has been implicated in neurodegenerative disorders, metabolic diseases, and cancer. Research approaches may include:

• Comparative analysis: Quantifying COX6A1 expression levels in normal versus pathological tissues using Western blot or IHC

• Co-localization studies: Combining COX6A1 antibody with markers of mitochondrial stress or quality control to assess association with pathological processes

• Functional correlation: Correlating COX6A1 expression levels with cytochrome c oxidase enzymatic activity measurements

• Mutation impact assessment: Studying how disease-associated mutations in mitochondrial or nuclear DNA affect COX6A1 incorporation into Complex IV

Researchers should consider that various pathological conditions may affect different aspects of COX assembly. For example, studies have shown that COX1-COX14-COA3-CMC1 complex (MITRAC) accumulates in several mutants affecting later steps of Complex IV assembly, such as in COX2 or COX3 homoplasmic mutant cybrids, or in cells with silenced COX4, COX5A, or COX10 .

What are common challenges in detecting COX6A1 and how can they be addressed?

Researchers may encounter several challenges when detecting COX6A1:

• Low signal intensity: Due to the small size (9.5 kDa) and potentially low expression levels of COX6A1, detection can be challenging. Solutions include:

  • Using higher antibody concentrations within the recommended range

  • Extending primary antibody incubation time (overnight at 4°C)

  • Employing more sensitive detection systems (ECL Prime or SuperSignal West Femto)

  • Increasing protein loading (up to 100 μg per lane)

• Non-specific bands: May occur particularly when using polyclonal antibodies. Potential solutions:

  • Optimize blocking conditions (try different blockers like 5% milk, 5% BSA, or commercial blockers)

  • Increase washing stringency (add 0.1-0.3% SDS to wash buffers)

  • Decrease primary antibody concentration

  • Consider using a monoclonal antibody for higher specificity

• Inconsistent results across tissues/cells: Different cell types may have varying levels of COX6A1 expression. Approach:

  • Refer to validated positive controls (mouse liver tissue, NIH/3T3 cells, rat liver tissue for Western blot; human pancreatic cancer tissue for IHC; MCF-7 and SH-SY5Y cells for IF/ICC)

  • Optimize protocols for specific sample types

How should researchers approach antigen retrieval optimization for COX6A1 immunohistochemistry?

Antigen retrieval is a critical step for successful COX6A1 detection in fixed tissues. The recommended approach includes:

• Primary recommendation: TE buffer at pH 9.0 has been validated for optimal COX6A1 epitope retrieval

• Alternative method: Citrate buffer at pH 6.0 can be used as an alternative, though potentially with reduced epitope accessibility

• Optimization strategy:

  • Test both buffer systems side by side

  • Vary retrieval times (10-30 minutes)

  • Compare different heating methods (microwave, pressure cooker, water bath)

  • Include positive control tissues (human pancreatic cancer tissue has been validated)

• Considerations for specific fixatives:

  • Formalin-fixed tissues typically require more aggressive retrieval

  • Fresh frozen sections may require gentler retrieval or none at all

  • Alcohol-fixed specimens often show better antigen preservation

A systematic approach to optimization will help determine the ideal antigen retrieval conditions for specific experimental settings.

How can COX6A1 antibody be used in studies of mitochondrial diseases and disorders?

COX6A1 antibody serves as an important tool for investigating mitochondrial diseases, particularly those affecting oxidative phosphorylation. Research applications include:

• Diagnostic markers: Assessment of COX6A1 expression or assembly into Complex IV may serve as a biomarker for certain mitochondrial disorders

• Pathophysiological studies: Investigation of how mutations in mitochondrial or nuclear DNA affect COX6A1 incorporation and Complex IV assembly

• Therapeutic response monitoring: Evaluation of mitochondrial function restoration in response to potential therapeutics

Researchers should note that diseases affecting different components of the respiratory chain may have distinct patterns of COX6A1 expression and incorporation. For example, studies have shown that COX1-COX14-COA3-CMC1 complex (MITRAC) accumulates in several mutants affecting steps of Complex IV assembly, but COX1 synthesis remains unaffected in these mutants .

What considerations are important when using COX6A1 antibody in multi-color immunofluorescence experiments?

Multi-color immunofluorescence allows co-localization studies of COX6A1 with other mitochondrial or cellular proteins. Key considerations include:

• Antibody host species compatibility: When combining multiple primary antibodies, they should be from different host species or isotypes to allow specific secondary antibody detection

• Fluorophore selection: Choose fluorophores with minimal spectral overlap to reduce bleed-through artifacts

• Sequential staining: For challenging combinations, consider sequential staining protocols rather than simultaneous incubation

• Controls:

  • Single-color controls to establish the specificity of each detection channel

  • Secondary-only controls to assess non-specific binding

  • Isotype controls to evaluate background staining

• Mitochondrial co-markers: Common proteins to co-stain with COX6A1 include:

  • TOMM20 (outer membrane)

  • Cytochrome c (intermembrane space)

  • COXIV (inner membrane)

  • HSP60 (matrix)

Successful multi-color immunofluorescence has been demonstrated with COX6A1 antibodies in cell lines such as MCF-7 and SH-SY5Y .

How can researchers effectively use COX6A1 antibody in flow cytometry applications?

Flow cytometry allows quantitative assessment of COX6A1 expression at the single-cell level. For effective flow cytometry experiments with COX6A1 antibody:

• Fixation and permeabilization: Both 80% methanol (5 min) and 4% paraformaldehyde followed by 0.1% PBS-Tween permeabilization (20 min) have been validated for COX6A1 detection

• Blocking: Use 10% normal goat serum with 0.3M glycine in PBS to reduce non-specific binding

• Antibody concentration: Typically 1 μg per 10^6 cells yields good results

• Controls:

  • Isotype control antibody (e.g., mouse IgG1 for the Abcam monoclonal antibody)

  • Unlabeled sample as additional negative control

  • Positive control cell line (HepG2 cells have been validated)

• Acquisition parameters:

  • Collect >5,000 events for reliable statistics

  • For Alexa Fluor 488-conjugated secondary antibodies, use a 488nm laser with a 525/30 bandpass filter

  • Compensation is critical when combining with other fluorescent markers

When analyzing results, consider that mitochondrial content and COX6A1 expression may vary significantly across cell types and physiological states.