ATP6-1 Antibody

What is ATP6-1 and why is it important in research?

ATP6 (ATP synthase subunit a-1) is a critical component of mitochondrial F0F1-ATP synthase, also known as Complex V. This enzyme complex synthesizes ATP from ADP and inorganic phosphate using the proton motive force created by respiratory electron transport. In Arabidopsis, ATP6 is encoded by multiple loci (ATMG00410/ATMG01170/AT2G07741) and plays an essential role in cellular energy production . The study of ATP6 and its mutations is crucial for understanding mitochondrial diseases, bioenergetics, and cellular metabolism. Antibodies targeting ATP6-1 enable researchers to detect, quantify, and characterize this protein in various experimental contexts.

How do ATP6-1 antibodies differ from MT-ATP6 antibodies used in human research?

While ATP6-1 antibodies are commonly used in plant research (especially in Arabidopsis and other plant species), MT-ATP6 antibodies specifically target the human mitochondrial ATP6 protein encoded by the MT-ATP6 gene. The human MT-ATP6 protein has a molecular weight of approximately 24,817 daltons . The key differences lie in species specificity and epitope recognition. Plant ATP6-1 antibodies typically recognize conserved regions in plant ATP synthase subunit a, while human MT-ATP6 antibodies target species-specific epitopes in the human protein. When selecting an antibody, researchers must consider cross-reactivity profiles based on their experimental model organism.

What is the relationship between ATP6-1, ATP6, and ATP synthase complex?

ATP6-1 is a specific isoform of the ATP6 protein, which functions as subunit a of the F0 portion of the ATP synthase complex. The complete mitochondrial F0F1-ATP synthase (Complex V) consists of multiple subunits organized into two main domains: the F1 catalytic domain and the F0 membrane domain. ATP6/ATP6-1 is an integral membrane protein within the F0 domain that forms part of the proton channel necessary for ATP synthesis . The proper assembly and function of ATP6 within this complex is essential for oxidative phosphorylation and cellular energy production.

What are the validated applications for ATP6-1 antibodies in research?

ATP6-1 antibodies have been validated for multiple applications, including:

The selection of application should be guided by the specific antibody's validation profile, as not all ATP6-1 antibodies perform equally across all techniques . Recent research has employed these antibodies in studying mitochondrial complex assembly and function, as demonstrated in a 2022 study on maize seed development .

How should researchers optimize Western blot protocols for ATP6-1 detection?

For optimal Western blot detection of ATP6-1:

• Sample preparation: Isolate mitochondria using differential centrifugation to enrich for mitochondrial proteins. Solubilize samples in a buffer containing 1-2% non-ionic detergent (e.g., Triton X-100 or digitonin).

• Gel electrophoresis: Use 10-15% SDS-PAGE gels for optimal resolution of ATP6 (approximately 25 kDa in most species).

• Transfer conditions: Transfer to PVDF membranes at 80-100V for 60-90 minutes in cold transfer buffer containing 10-20% methanol.

• Blocking: Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute ATP6-1 antibody 1:1000-1:5000 in blocking buffer and incubate overnight at 4°C.

• Detection: Use HRP-conjugated secondary antibodies and enhanced chemiluminescence for visualization.

• Controls: Include positive controls (purified ATP synthase complex) and negative controls (ATP6 knockout samples when available) .

What cross-reactivity considerations should researchers be aware of when using ATP6-1 antibodies?

ATP6-1 antibodies show varying degrees of cross-reactivity across species. For instance, antibodies raised against peptide sequences from Arabidopsis ATP6-1 demonstrate cross-reactivity with homologous proteins from multiple plant species including Solanum tuberosum, Nicotiana tabacum, Brassica species, Oryza sativa, and Zea mays . The high degree of conservation in ATP synthase subunits explains this cross-reactivity pattern.

Researchers should note that some ATP6-1 antibodies may not distinguish between ATP6-1 and ATP6-2 isoforms due to sequence homology. For example, the synthetic peptide immunogen used in one commercial antibody shows 100% (15/15) homology with sequences in both ATP6-1 (AT2G07741) and ATP6-2 (ATMG01170) . When isoform specificity is crucial, researchers should select antibodies raised against unique regions or validate specificity using knockout/knockdown controls.

How are ATP6-1 antibodies used to investigate pathogenic mutations in ATP6?

ATP6-1 antibodies serve as valuable tools for investigating the effects of pathogenic mutations in ATP6 genes. In human research, antibodies have been used to study mutations like m.9191T>C, which converts a highly conserved leucine residue to proline in ATP synthase subunit a . Similar approaches can be applied to plant models.

For such studies, researchers typically:

• Generate or identify mutant cell lines/organisms carrying specific ATP6 mutations

• Use ATP6-1 antibodies to assess protein expression, stability, and localization

• Compare wild-type and mutant samples to determine how mutations affect protein incorporation into the ATP synthase complex

• Correlate biochemical findings with functional outcomes (ATP synthesis rate, growth defects, etc.)

The table below summarizes findings from research on leucine-to-proline mutations in ATP6:

These studies demonstrate how antibody-based approaches help elucidate the molecular consequences of ATP6 mutations .

What role do ATP6-related proteins play in cancer research, and how are antibodies used to study them?

Recent research has highlighted the potential role of ATP6-related proteins in cancer biology. For example, ATP6AP1 (ATP6 accessory protein 1) has been identified as significantly upregulated in colorectal cancer (CRC) and associated with poor clinicopathological characteristics and prognosis .

Antibodies against ATP6 and related proteins enable researchers to:

• Assess expression levels in tumor versus normal tissues

• Correlate expression with clinical outcomes and tumor characteristics

• Investigate interactions between ATP6-related proteins and immune cell infiltration

• Evaluate potential as diagnostic or prognostic biomarkers

How can researchers use ATP6-1 antibodies to study mitochondrial complex assembly?

ATP6-1 antibodies are valuable tools for investigating the assembly of ATP synthase and its integration with other mitochondrial complexes. A 2022 study demonstrated that an MCIA-like complex is required for mitochondrial complex I assembly and seed development in maize .

To study complex assembly, researchers can employ:

• Blue Native PAGE combined with ATP6-1 antibody detection to visualize intact complexes

• Co-immunoprecipitation with ATP6-1 antibodies to identify interaction partners

• Immunofluorescence microscopy to assess co-localization with other complex components

• Time-course analysis with ATP6-1 antibodies to track assembly dynamics during development

These approaches help elucidate how ATP6-1 incorporates into the ATP synthase complex and how defects in this process may contribute to mitochondrial dysfunction in various biological contexts.

What are the most common technical challenges when working with ATP6-1 antibodies?

Researchers commonly encounter several technical challenges when working with ATP6-1 antibodies:

• Background signal: Due to the hydrophobic nature of ATP6 as a membrane protein, nonspecific binding can occur. Optimize blocking conditions (try 5% BSA instead of milk for membrane proteins) and include 0.1-0.3% Triton X-100 in antibody diluents.

• Multiple bands: ATP6 may show multiple bands due to processing, degradation, or cross-reactivity. To address this:

  • Include protease inhibitors during sample preparation

  • Use freshly prepared samples

  • Validate bands using knockout/knockdown controls

  • Consider pre-absorbing the antibody with non-specific proteins

• Weak signal: ATP6 is often expressed at moderate levels. Enhance detection by:

  • Enriching for mitochondrial fractions

  • Using signal amplification systems

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

  • Testing multiple antibody clones for optimal sensitivity

How should researchers interpret discrepancies between ATP6-1 antibody results and transcriptomic data?

Discrepancies between protein detection (using ATP6-1 antibodies) and mRNA expression (from transcriptomic data) are not uncommon and may result from:

• Post-transcriptional regulation: mRNA levels may not directly correlate with protein abundance due to translational efficiency, protein stability, or degradation rates.

• Post-translational modifications: Some antibodies may detect only specific modified forms of ATP6-1, leading to apparent discrepancies with total transcript levels.

• Protein localization or extraction efficiency: Membrane proteins like ATP6-1 may be inefficiently extracted in certain buffers, resulting in lower detection despite high transcript levels.

• Antibody specificity issues: Cross-reactivity or epitope masking may affect antibody binding and detection.

To resolve such discrepancies:

• Validate findings using multiple antibodies targeting different epitopes

• Employ orthogonal protein detection methods (mass spectrometry)

• Use tagged versions of the protein (if feasible in your system)

• Investigate protein turnover rates and post-translational modifications

What considerations should researchers take when designing experiments to study ATP6-1 mutations?

When designing experiments to study ATP6-1 mutations, researchers should consider:

• Mutation selection strategy:

  • Target evolutionarily conserved residues for maximum impact

  • Consider known pathogenic mutations in homologous proteins

  • Use structural information to predict functional consequences

• Model system selection:

  • Yeast models permit efficient mitochondrial transformation

  • Plant systems may require specialized transformation approaches

  • Cell culture models may be appropriate for human ATP6 studies

• Functional assays:

  • ATP synthesis rate measurement using luciferase-based assays

  • Oxygen consumption rate determination

  • Membrane potential measurement using fluorescent dyes

  • Growth assays on fermentable versus non-fermentable carbon sources

• Protein analysis approaches:

  • Blue Native PAGE to assess complex assembly

  • Antibody-based detection of expression levels and localization

  • Pulse-chase experiments to assess protein stability

  • Cross-linking studies to investigate protein-protein interactions

How are ATP6-1 antibodies being used in plant stress response research?

ATP6-1 antibodies are increasingly employed in studying how plant mitochondrial function adapts during environmental stress. Research indicates that ATP synthase composition and activity can be modulated in response to drought, salt stress, temperature extremes, and pathogen attack.

Methodological approaches include:

• Comparing ATP6-1 protein levels across stress conditions using quantitative Western blotting

• Assessing ATP synthase complex integrity under stress using Blue Native PAGE followed by immunodetection

• Investigating post-translational modifications of ATP6-1 during stress responses

• Correlating ATP6-1 expression with ATP production capacity in stressed tissues

These approaches help elucidate how plants regulate energy metabolism during environmental challenges, which has implications for crop improvement strategies .

What new methodologies are being developed for studying ATP6-1 interaction partners?

Advanced methodologies for studying ATP6-1 interaction partners include:

• Proximity-dependent biotin labeling (BioID or TurboID): By fusing a biotin ligase to ATP6-1, researchers can identify proximal proteins in the native cellular environment, which is particularly valuable for membrane protein complexes.

• Cross-linking mass spectrometry (XL-MS): Chemical cross-linking followed by mass spectrometry analysis allows identification of proteins in direct contact with ATP6-1 and can provide structural insights into protein complexes.

• Cryo-electron microscopy: Combined with antibody-based labeling, cryo-EM provides structural information about ATP6-1 within the ATP synthase complex at near-atomic resolution.

• Split-GFP complementation: By tagging ATP6-1 and potential interaction partners with complementary GFP fragments, researchers can visualize interactions in living cells.

These emerging techniques complement traditional co-immunoprecipitation approaches and provide more comprehensive insights into ATP6-1 interactions within the complex mitochondrial environment .

How might ATP6-1 antibodies contribute to biomarker development for mitochondrial diseases?

Research on ATP6 and related proteins suggests potential applications in biomarker development:

• Diagnostic applications: Antibodies against ATP6 and associated proteins could help identify mitochondrial dysfunction in patient samples. For instance, altered ATP6 expression, localization, or post-translational modifications might serve as indicators of mitochondrial disease.

• Prognostic indicators: As demonstrated with ATP6AP1 in colorectal cancer, where high expression correlates with poor prognosis , similar approaches could be applied to other conditions involving mitochondrial dysfunction.

• Treatment response monitoring: Antibody-based assays could track changes in ATP6 and related proteins during treatment interventions targeting mitochondrial function.

• Personalized medicine approaches: Characterizing ATP6 mutations and their effects on protein expression using specific antibodies could help guide treatment decisions for patients with mitochondrial diseases.

The development of standardized, validated antibody-based assays would be essential for translating these research applications into clinical utilities.