ATP6-2 Antibody

What is ATP6 and why is it significant in mitochondrial research?

ATP6, also known as MT-ATP6, mitochondrially encoded ATP synthase 6, ATPase6, or MTATP6, is a crucial protein subunit of mitochondrial ATP synthase (Complex V). This protein functions as part of the Fo region, specifically as the "a" subunit within the proton channel embedded in the inner mitochondrial membrane. ATP6 has a molecular weight of approximately 24.8 kilodaltons and plays a fundamental role in the regulation of mitochondrial ATP production and maintenance of mitochondrial membrane potential .

The significance of ATP6 in research extends beyond basic mitochondrial function, as pathogenic variants in the MT-ATP6 gene were among the first described human mitochondrial DNA diseases . These variants can lead to severe multi-system disorders including Leigh syndrome, NARP (neuropathy, ataxia, and retinitis pigmentosa), stroke, and cardiomyopathy, making ATP6 an important target for investigating mitochondrial disease mechanisms .

What experimental applications are ATP6 antibodies suitable for?

ATP6 antibodies have demonstrated utility across multiple experimental platforms:

For Western blot applications, ATP6 antibodies have been validated with various samples including mouse brain tissue, C2C12 cells, and rat brain . The observed molecular weight typically falls between 25-30 kDa, which aligns with the calculated molecular weight of 25 kDa . When designing experiments, researchers should optimize antibody dilutions for their specific sample types and detection systems.

How should researchers distinguish between different ATP6 antibody options?

When selecting an ATP6 antibody, researchers should consider the following criteria:

• Reactivity spectrum: Verify which species the antibody recognizes (human, mouse, rat, or others). For example, some antibodies like 55313-1-AP from Proteintech show reactivity with human, mouse, and rat samples .

• Clonality and host species: Determine whether a polyclonal or monoclonal antibody better suits your experimental needs. Polyclonal antibodies (such as rabbit anti-ATP6) offer higher sensitivity but potentially lower specificity compared to monoclonals.

• Validated applications: Confirm the antibody has been verified for your intended application. For instance, the Proteintech 55313-1-AP antibody has been validated for WB, IHC, IF, and ELISA applications .

• Immunogen information: Review the immunogen used to generate the antibody, as this affects epitope recognition. Peptide-derived antibodies may recognize different regions than those raised against recombinant proteins.

• Published citations: The number of publications using a particular antibody can indicate reliability. For example, the 55313-1-AP antibody has been cited in 54 publications for Western blot applications alone .

How do heteroplasmy levels of MT-ATP6 variants correlate with clinical phenotypes?

Heteroplasmy levels (the percentage of mitochondrial DNA molecules carrying a mutation) in MT-ATP6 variants demonstrate significant correlation with both symptom presence and disease severity. Meta-analysis of reported cases reveals:

• Symptomatic vs. asymptomatic carriers: Despite some overlap at the individual level, symptomatic patients exhibit significantly higher heteroplasmy loads compared to asymptomatic relatives (p=3.2×10^−45) .

• Phenotype severity correlation: Higher heteroplasmy levels correlate with more severe clinical presentations. Patients with early-onset Leigh syndrome demonstrate significantly higher heteroplasmy levels compared to those with later-onset NARP syndrome (p=0.037) .

• Age of onset correlation: A negative correlation exists between heteroplasmy level and age of onset (Pearson correlation coefficient=−0.37, p=1.6×10^−7), indicating that higher mutation loads typically result in earlier disease manifestation .

When designing experiments investigating MT-ATP6 variants, researchers should quantify heteroplasmy levels in multiple tissues when possible, as levels may vary between tissues and affect interpretation of pathogenicity.

What biochemical complexities arise when characterizing MT-ATP6 variants?

The biochemical characterization of MT-ATP6 variants presents several methodological challenges:

• Heterogeneous biochemical findings: No universal biochemical signature exists across all MT-ATP6 pathogenic variants. The most common observations include reduced ATP synthesis rate, preserved ATP hydrolysis capacity, and abnormally increased mitochondrial membrane potential, but these findings are inconsistent across variants .

• Lack of standardized functional assays: Despite the clinical importance of MT-ATP6 variants, there remains a lack of clinically-available functional assays, complicating the validation of variant pathogenicity .

• Variant classification challenges: The absence of consistent biochemical markers makes accurate classification of variants of uncertain significance (VUS) particularly difficult for MT-ATP6 .

When investigating novel MT-ATP6 variants, researchers should employ multiple complementary biochemical approaches rather than relying on a single assay, as no individual test has proven universally diagnostic.

What are the optimal Western blot conditions for detecting ATP6?

Achieving optimal Western blot results for ATP6 detection requires attention to several methodological details:

• Sample preparation: For mitochondrial proteins like ATP6, enrichment of the mitochondrial fraction often improves detection. Gentle lysis conditions help preserve protein integrity.

• Protein loading: Load 20-40μg of total protein per lane when using whole cell/tissue lysates, or 10-15μg when using enriched mitochondrial fractions.

• Gel percentage: 12-15% SDS-PAGE gels are recommended for optimal resolution of ATP6 (25-30 kDa).

• Transfer conditions: Semi-dry transfer (15-20V for 30-45 minutes) or wet transfer (30V overnight at 4°C) are both effective for ATP6.

• Blocking and antibody dilution: 5% non-fat milk in TBST is typically effective for blocking. For primary antibody incubation, a dilution of 1:500-1:1000 is recommended for many commercial ATP6 antibodies .

• Detection system optimization: Enhanced chemiluminescence (ECL) detection systems with medium sensitivity are generally sufficient for ATP6 detection in most sample types.

How can researchers differentiate between pathogenic and non-pathogenic MT-ATP6 variants?

Distinguishing pathogenic from benign MT-ATP6 variants requires integration of multiple lines of evidence:

• Heteroplasmy quantification: Higher heteroplasmy levels correlate significantly with disease presence and severity, though there is overlap between symptomatic and asymptomatic individuals .

• Functional studies: Assessment of ATP synthesis rates, ATP hydrolysis capacity, and mitochondrial membrane potential can provide evidence of functional impairment, though findings vary across variants .

• Conservation analysis: Evaluate evolutionary conservation of the affected amino acid position across species.

• Family segregation studies: Track variant segregation with disease phenotype across multiple family members when possible.

• Population frequency data: Pathogenic variants are typically absent or extremely rare in population databases.

A multi-faceted approach combining these methods provides the strongest evidence for variant pathogenicity classification, as no single method has proven definitive for MT-ATP6 variants.

What controls should be included when working with ATP6 antibodies?

Proper experimental controls are essential for reliable interpretation of ATP6 antibody results:

• Positive tissue/cell controls: Include samples known to express ATP6, such as mouse brain tissue, C2C12 cells, or rat brain tissue, which have been validated with commercial antibodies .

• Loading controls: For Western blot applications, include mitochondrial matrix proteins (e.g., HSP60) or other mitochondrial membrane proteins (e.g., VDAC) as loading controls rather than typical cytosolic housekeeping proteins.

• Negative controls: When possible, include ATP6-depleted samples or samples from models with reduced ATP6 expression.

• Antibody validation controls: Consider using blocking peptides specific to the immunogen used to generate the antibody to confirm specificity in critical experiments.

• Cross-species validation: When working with non-human models, verify antibody cross-reactivity with the target species before conducting full experiments.

How should researchers interpret contradictory results between different biochemical assays for MT-ATP6 variants?

The literature demonstrates significant variability in biochemical findings across different MT-ATP6 variants and testing methods. When encountering contradictory results:

• Consider variant-specific effects: Different MT-ATP6 variants may disrupt distinct aspects of ATP synthase function, resulting in variability across biochemical assays .

• Evaluate assay sensitivity: Some assays may be more sensitive to subtle functional changes than others.

• Assess tissue-specific effects: Results may vary between different tissue types due to differences in mitochondrial content, heteroplasmy levels, or compensatory mechanisms.

• Replicate findings using multiple methods: Whenever possible, confirm results using alternative approaches that measure the same parameter through different mechanisms.

• Integrate with clinical and genetic data: Interpret biochemical findings in the context of patient phenotype and heteroplasmy level, as these correlations are more consistently observed than specific biochemical signatures .

What novel approaches are being developed to assess MT-ATP6 variant pathogenicity?

The challenges in establishing clear biochemical markers for MT-ATP6 variants have prompted development of new methodological approaches:

• High-resolution respirometry: More sensitive methods to detect subtle changes in mitochondrial respiratory function are being applied to MT-ATP6 variant characterization.

• Live-cell imaging techniques: Advanced microscopy methods to visualize mitochondrial membrane potential and ATP production in real-time may provide more nuanced functional data.

• Patient-derived cellular models: Induced pluripotent stem cell (iPSC) technologies allow generation of patient-specific cell models that maintain original heteroplasmy levels.

• CRISPR-based mitochondrial genome editing: Emerging techniques for precise mtDNA editing offer potential for creating isogenic cell lines differing only in MT-ATP6 sequence.

• Computational modeling: Structure-based prediction of variant effects on ATP synthase function is improving as more structural data becomes available.

These emerging approaches may provide more consistent methods for determining the pathogenicity of MT-ATP6 variants and addressing the current limitations in variant classification.