COX17-2 Antibody

What is COX17 and what is its role in mitochondrial function?

COX17 functions as a copper chaperone and assembly factor for mitochondrial electron transport chain complex IV (cytochrome c oxidase). It plays a critical role in maintaining mitochondrial structure and function. Research indicates that COX17 is essential for the establishment and maintenance of inner mitochondrial membrane architecture . Importantly, COX17 facilitates copper delivery to downstream proteins in the electron transport chain, with mutations in the copper-binding domains (e.g., C22,23A) resulting in loss of function .

Methodologically, when studying COX17 function, researchers should consider:

• Evaluating mitochondrial morphology (fragmentation patterns) using fluorescence microscopy

• Assessing cristae density through transmission electron microscopy (TEM)

• Measuring mitochondrial membrane potential with potential-sensitive dyes

• Quantifying complex IV activity with standard respiratory chain enzyme assays

How does COX17 differ from COX-2 in terms of structure and function?

COX17 and COX-2 are fundamentally different proteins with distinct cellular functions:

Understanding these differences is crucial when selecting antibodies for research, as cross-reactivity between these proteins is unlikely but should be verified .

What are the key considerations when selecting antibodies against COX17 or COX-2?

When selecting antibodies for COX17 or COX-2 research, consider:

For COX17 antibodies:

• Epitope specificity: Determine whether the antibody recognizes specific domains, particularly if studying acetylated forms (K18, K30)

• Species cross-reactivity: Human COX17 has sequence conservation with mouse COX17

• Application compatibility: Verify validation for your specific application (western blot, immunofluorescence, etc.)

For COX-2 antibodies:

• Specificity: Confirm the antibody does not cross-react with COX-1, as demonstrated for clones like AS67

• Detection method: Consider fluorophore conjugation (e.g., PE conjugation) for flow cytometry applications

• Validation status: Review published validation methods including ELISA confirmation of specificity

How can researchers validate COX17 or COX-2 antibody specificity?

Methodological approaches to validate antibody specificity:

For COX17:

• Knockdown/knockout validation: Use COX17 shRNA or CRISPR-Cas9 knockout cells as negative controls

• Overexpression systems: Express tagged COX17 variants (wild-type, K18,30Q, K18,30R) to confirm antibody detection patterns

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Western blot analysis comparing endogenous and exogenous protein levels

For COX-2:

• ELISA testing against recombinant COX-1 and COX-2 proteins to confirm specificity

• Parallel testing with multiple antibody clones (e.g., AS67) targeting different epitopes

• Induction experiments: Test antibody detection in samples with known COX-2 induction versus baseline

What are optimal conditions for detecting acetylated forms of COX17?

Detecting acetylated COX17 requires specific methodological considerations:

• Antibody selection: Use antibodies specifically validated for acetyl-lysine detection at positions K18 and K30 of COX17

• Sample preparation:

  • Include deacetylase inhibitors (e.g., nicotinamide, trichostatin A) in lysis buffers

  • Maintain cold temperatures throughout processing to preserve acetylation

• Controls:

  • Include K18,30Q (acetylation-mimetic) and K18,30R (non-acetylatable) COX17 variants as controls

  • Use MOF-depleted cells as a negative control for acetylation

• Validation approach:

  • Use mass spectrometry to confirm acetylation sites with adjusted P value <0.01

  • Perform parallel western blot with pan-acetyl-lysine and COX17-specific antibodies

How can researchers troubleshoot issues with COX-2 antibody staining?

When encountering problems with COX-2 antibody staining:

• Fixation optimization:

  • For flow cytometry: Test different permeabilization methods compatible with PE-conjugated antibodies

  • For IHC/IF: Optimize fixative type and duration

• Blocking parameters:

  • Ensure appropriate blocking to reduce non-specific binding

  • Consider protein-free blockers if background persists

• Antibody concentration:

  • Perform titration experiments to determine optimal concentration

  • Include isotype controls at matching concentrations

• Storage considerations:

  • Store conjugated antibodies at 2-8°C without freezing

  • Protect fluorophore-conjugated antibodies from light exposure

• Signal amplification:

  • Consider biotin-streptavidin systems for weak signals

  • Evaluate alternative detection methods if needed

How can researchers study the interaction between COX17 acetylation and mitochondrial function?

To investigate the relationship between COX17 acetylation and mitochondrial function:

• Generate stable cell lines expressing:

  • Wild-type COX17 (hCOX17 WT)

  • Acetylation-mimicking variant (hCOX17 K18,30Q)

  • Non-acetylatable variant (hCOX17 K18,30R)

• Simultaneously deplete endogenous COX17 using shRNA to observe the full impact of acetylation mutants

• Assess mitochondrial parameters:

  • Mitochondrial morphology using fluorescence microscopy

  • Cristae density via transmission electron microscopy

  • Membrane potential using potential-sensitive dyes

  • Complex IV activity with respiratory chain enzyme assays

• Compare rescue capabilities:

  • Wild-type COX17 should partially rescue defects

  • K18,30Q variant should rescue most defects even in the absence of MOF

  • K18,30R variant should fail to rescue phenotypes

• Measure secondary effects:

  • Evaluate mitochondrial ROS production

  • Assess protein turnover rates of different COX17 variants

  • Analyze transcriptional responses using RNA-seq

What approaches can be used to study anti-COX-2 autoantibodies in disease states?

For investigating anti-COX-2 autoantibodies in diseases like immune aplastic anemia (IAA):

• Detection methods:

  • Protein microarray screening (covering >9000 proteins) for initial discovery

  • ELISA validation in large cohorts (e.g., 405 patients vs. 815 controls)

• Clinical correlation analysis:

  • Evaluate relationships between antibody positivity and clinical parameters (e.g., platelet counts)

  • Assess correlations with age and genetic factors (e.g., HLA-DRB1*15:01 genotype)

• Specificity determination:

  • Test against related diseases and healthy controls

  • Evaluate sensitivity in specific patient subgroups (e.g., 83% in >40 years old IAA patients with HLA-DRB1*15:01)

• Functional studies:

  • Investigate mechanisms by which autoantibodies affect target cells

  • Test passive transfer of purified antibodies in experimental models

What controls should be included when studying COX17 function?

When designing experiments to study COX17 function:

Essential controls include:

• Genetic controls:

  • COX17 knockdown (shRNA) or knockout cells

  • Rescue experiments with wild-type COX17

  • Functional mutants: K18,30Q (acetylation-mimetic), K18,30R (non-acetylatable), and C22,23A (copper binding-deficient)

• Pathway controls:

  • MOF knockdown cells (upstream regulator of COX17 acetylation)

  • MOF-KANSL complex members knockdown

  • Mitochondrially-targeted MOF for rescue experiments

• Functional readouts:

  • Mitochondrial morphology assessments

  • Membrane potential measurements

  • Complex IV activity assays

  • Alternative oxidase (AOX) expression to bypass complex IV defects

• Secondary effect controls:

  • Assessment of other respiratory complexes (I, II, III, V)

  • Evaluation of fusion proteins (e.g., MFN1)

  • Analysis of cardiolipin species

How should researchers design experiments to differentiate between COX-1 and COX-2?

To properly differentiate between COX-1 and COX-2 in experimental settings:

• Antibody selection:

  • Use validated antibodies with confirmed specificity (e.g., clone AS67 for COX-2 and AS70 for COX-1)

  • Verify lack of cross-reactivity by ELISA with recombinant proteins

• Expression pattern validation:

  • COX-1 is constitutively expressed

  • COX-2 is inducible and can be triggered with appropriate stimuli

• Subcellular localization:

  • Both are found on the lumenal surface of the endoplasmic reticulum and on inner/outer membranes of the nuclear envelope

  • Use co-localization studies with organelle markers

• Multicolor flow cytometry approach:

  • Use dual staining with differently conjugated antibodies (e.g., COX-1 FITC/COX-2 PE)

  • Include appropriate compensation controls

  • Gate based on known expression patterns in relevant cell types

How can COX17 research contribute to understanding mitochondrial diseases?

COX17 research has significant implications for mitochondrial diseases:

• Patient-derived models:

  • Fibroblasts from patients with MOF syndrome show respiratory defects that can be restored by:

    • Alternative oxidase (AOX) expression

    • Acetylation-mimetic COX17 (K18,30Q)

    • Mitochondrially-targeted MOF

• Therapeutic approach exploration:

  • Expression of acetylation-mimetic COX17 (K18,30Q) can rescue complex IV activity in patient cells

  • This suggests potential for targeted therapies that enhance COX17 acetylation or bypass its dysfunction

• Diagnostic applications:

  • Evaluation of complex IV activity

  • Assessment of mitochondrial morphology

  • Measurement of COX17 acetylation status

• Disease mechanism insights:

  • RNA-seq analysis of patient cells reveals that alternative oxidase (AOX) expression leads to transcriptional changes that partially restore normal gene expression patterns

What is the significance of anti-COX-2 autoantibodies as biomarkers in clinical research?

Anti-COX-2 autoantibodies represent important biomarkers with clinical significance:

• Diagnostic value:

  • 37% of adult immune aplastic anemia (IAA) patients test positive for anti-COX-2 antibodies

  • Only 1.7% of controls show positivity

• Subgroup identification:

  • Anti-COX-2 antibodies define a distinct IAA subgroup

  • These patients present with lower platelet counts

• Correlation with patient characteristics:

  • Positivity correlates with age and HLA-DRB1*15:01 genotype

  • 83% of IAA patients >40 years with HLA-DRB1*15:01 test positive

• Disease specificity:

  • Sporadic positive cases observed in related bone marrow failure diseases, multiple sclerosis, and type I diabetes

  • No positivity in healthy controls or patients with non-autoinflammatory diseases or rheumatoid arthritis

• Research applications:

  • Can serve as inclusion criteria for clinical studies

  • May help stratify patients for targeted therapeutic approaches

  • Provides insight into autoimmune mechanisms in bone marrow failure