ATP9 Antibody

What is ATP9 and why is it significant in mitochondrial research?

ATP9 (also called subunit 9) is a critical component of the mitochondrial ATP synthase, functioning as part of the membrane-embedded F₀ sector that facilitates proton transport. Its significance stems from its essential role in oxidative phosphorylation and cellular energy production. In organisms like Saccharomyces cerevisiae (baker's yeast), ATP9 is typically encoded by the mitochondrial genome, though in many other organisms including filamentous fungi and most animals, the gene has been relocated to the nuclear genome . The protein forms a ring structure within the F₀ complex that is crucial for the rotary mechanism of ATP synthesis. Research involving ATP9 antibodies has been instrumental in understanding mitochondrial biogenesis, ATP synthase assembly, and evolutionary relocation of mitochondrial genes to the nucleus, making it a focal point for investigations into mitochondrial disorders and energy metabolism .

How do ATP9 antibodies differ from ATP9A antibodies?

It is critical for researchers to distinguish between antibodies targeting mitochondrial ATP9 (subunit 9 of ATP synthase) and those targeting ATP9A, which is a different protein entirely. ATP9A belongs to the P4-ATPase family of phospholipid flippases and primarily localizes to endosomes, playing important roles in endosomal recycling pathways . While mitochondrial ATP9 antibodies are used to study ATP synthase assembly and mitochondrial function, ATP9A antibodies are employed in research related to membrane trafficking, neurodevelopment, and neurological disorders such as ADHD . When selecting or developing antibodies, researchers must verify target specificity through appropriate controls to avoid cross-reactivity between these distinct proteins, as this can lead to misinterpretation of experimental results and confusion in the literature.

What are the recommended methods for validating ATP9 antibody specificity?

Validating ATP9 antibody specificity requires a multi-tiered approach:

• Knockout/knockdown controls: Test antibody reactivity in samples where ATP9 expression has been eliminated (e.g., using CRISPR-Cas9) or reduced (e.g., via siRNA). The Δatp9 yeast strain described in the literature provides an excellent negative control for mitochondrial ATP9 antibodies .

• Overexpression systems: Compare reactivity in samples with native versus overexpressed ATP9 protein. The allotopic expression systems described in the literature, where nuclear versions of ATP9 are expressed in mitochondrial ATP9-deficient backgrounds, can serve as positive controls .

• Cross-species reactivity analysis: Test the antibody against ATP9 proteins from different species to determine conservation of the epitope recognition. This is particularly important given the evolutionary relocation of ATP9 from mitochondrial to nuclear genomes across species .

• Western blotting with subcellular fractionation: Compare antibody reactivity in mitochondrial versus non-mitochondrial fractions to confirm specific detection of mitochondrial ATP9 .

• Peptide competition assays: Pre-incubate the antibody with excess purified ATP9 peptide or protein before using it in your application to confirm specific binding.

How can ATP9 antibodies be used to track mitochondrial-to-nuclear gene transfer experiments?

For researchers investigating the evolutionary phenomenon of mitochondrial-to-nuclear gene transfer, ATP9 antibodies serve as powerful tools to track the success of experimental gene relocation:

• Monitoring protein import and processing: When relocating ATP9 from the mitochondrial to nuclear genome, researchers can use ATP9 antibodies to detect whether the nuclear-encoded protein (with attached mitochondrial targeting sequence) is properly imported into mitochondria and processed to its mature form. In successful relocations, Western blotting will reveal both the precursor and mature forms of the protein in mitochondrial fractions .

• Assessing assembly efficiency: ATP9 antibodies can be employed in blue native PAGE and immunoprecipitation experiments to determine whether the nuclear-encoded ATP9 assembles properly into the ATP synthase complex. This is critical because relocation often requires adaptations to ensure proper assembly .

• Quantifying expression levels: Through quantitative Western blotting using ATP9 antibodies, researchers can compare expression levels between native mitochondrial-encoded ATP9 and experimental nuclear-encoded versions to optimize expression constructs .

• Detecting degradation products: ATP9 antibodies can identify degradation intermediates when nuclear-encoded ATP9 fails to properly import or assemble, helping researchers diagnose problems in their relocation strategies. For example, the detection of unprocessed yAtp9-Nuc in yeast mitochondria indicated a failure to cross the inner membrane .

• Visualizing subcellular localization: Through immunofluorescence microscopy with ATP9 antibodies, researchers can confirm the mitochondrial localization of relocated ATP9 proteins and identify mislocalization issues.

What methodological approaches should be used when studying ATP9 in the context of mitochondrial disease models?

When investigating mitochondrial diseases using ATP9 antibodies, researchers should employ these methodological approaches:

• Tissue-specific expression analysis: As mitochondrial diseases often affect tissues differently, use ATP9 antibodies to quantify ATP9 levels across multiple tissue types in disease models. This should include:

  • Western blot analysis of tissue lysates with appropriate loading controls

  • Immunohistochemistry to visualize spatial distribution within tissues

  • Flow cytometry for cell-type specific quantification

• Assembly state assessment: Since proper ATP synthase assembly is critical for function:

  • Use blue native PAGE followed by Western blotting with ATP9 antibodies to assess incorporation into ATP synthase complexes

  • Conduct co-immunoprecipitation with other ATP synthase subunits to determine interaction partners

  • Apply Förster resonance energy transfer (FRET) to study proximity to other subunits in living cells

• Functional correlation studies: To connect ATP9 abnormalities with disease phenotypes:

  • Measure ATP synthesis rates in parallel with ATP9 antibody staining intensity

  • Correlate ATP9 levels with membrane potential measurements

  • Assess the relationship between ATP9 expression patterns and markers of mitochondrial stress

• Mutation-specific antibodies: For known disease-causing mutations:

  • Develop or obtain antibodies that specifically recognize mutant forms of ATP9

  • Use these to distinguish between wild-type and mutant protein in heteroplasmic conditions

• Therapeutic monitoring: When testing potential therapies:

  • Track changes in ATP9 expression, localization, and assembly using antibodies

  • Establish baseline measurements before intervention and monitor throughout treatment

This multi-dimensional approach provides a comprehensive understanding of ATP9's role in disease pathogenesis and potential therapeutic responses.

How can ATP9 antibodies be utilized to study the interaction between ATP synthase assembly and translation regulation?

ATP9 antibodies provide valuable tools for investigating the complex relationship between ATP synthase assembly and translation regulation:

• Pulse-chase experiments: Use ATP9 antibodies in conjunction with metabolic labeling to track newly synthesized ATP9 and its assembly kinetics. This approach has revealed that translation of ATP synthase components like ATP9 can be regulated by the assembly state of the complex .

• Ribosome profiling: Combine ribosome profiling with ATP9 immunoprecipitation to identify ribosome-associated factors that may regulate ATP9 translation in response to assembly feedback. This method can help detect translational pauses or regulatory points during synthesis.

• Assembly-dependent translation analysis: In systems where mutations affect ATP synthase assembly, ATP9 antibodies can be used to determine whether translation of other subunits is affected. For example, research has shown that the L173P substitution in subunit 6 did not stimulate subunit 6 synthesis in mutants expressing nucleus-encoded ATP9, indicating that subunit 9 is required for upregulating subunit 6 in response to assembly defects .

• Comparative analysis of native versus nucleus-encoded ATP9: In experiments where both mitochondria-encoded and nucleus-encoded ATP9 are present, use specific antibodies to distinguish between them and determine their relative incorporation into functional complexes. This enables assessment of whether the source of ATP9 affects the assembly-translation feedback loop .

• Co-immunoprecipitation with translation factors: Use ATP9 antibodies to identify interactions with mitochondrial translation machinery components, potentially revealing direct links between the protein and translational regulation.

This multi-faceted approach allows researchers to decipher the intricate regulatory networks connecting ATP synthase assembly and the translation of its components.

What are the common pitfalls when using ATP9 antibodies in mitochondrial fractionation experiments?

When working with ATP9 antibodies in mitochondrial fractionation experiments, researchers should be aware of these common pitfalls and their solutions:

The proper detection of ATP9 often requires specific solubilization conditions, as demonstrated in studies where 2% digitonin was used for effective extraction of mitochondrial membrane proteins before SDS-PAGE analysis .

How should researchers approach epitope selection when developing ATP9 antibodies for different experimental applications?

The selection of appropriate epitopes for ATP9 antibody development requires careful consideration based on the intended experimental application:

• For detecting total ATP9 (regardless of assembly state):

  • Target conserved regions in the C-terminal domain that are accessible in both assembled and unassembled states

  • Avoid transmembrane domains which may be inaccessible in native conformations

  • Consider epitopes that remain exposed after processing of the mitochondrial targeting sequence

• For assembly-specific detection:

  • Target epitopes that become hidden or exposed during assembly into the ATP synthase complex

  • Develop antibodies against conformational epitopes that exist only in the assembled ring structure

  • Use structural data from cryo-EM studies to identify interface regions between ATP9 and other subunits

• For distinguishing between species variants:

  • Compare sequences between species (e.g., yeast ATP9 vs. Podospora anserina ATP9) and target divergent regions

  • This approach is particularly valuable when studying allotopic expression systems where foreign ATP9 proteins are expressed in yeast

• For detecting nucleus-encoded versus mitochondria-encoded ATP9:

  • Target epitopes in the mitochondrial targeting sequence present only in nucleus-encoded versions

  • Develop antibodies against regions modified by codon optimization in nucleus-encoded constructs

  • Consider using tagged versions with epitope tags when direct discrimination is challenging

• For detecting mutant forms:

  • Develop antibodies that specifically recognize disease-associated mutations

  • Target conformational changes induced by mutations that affect protein folding or stability

When developing new ATP9 antibodies, researchers should validate them using multiple approaches, including testing on samples from ATP9-null backgrounds like the Δatp9 yeast strain described in the literature .

What optimization strategies should be employed when using ATP9 antibodies in complex tissue samples?

Optimizing ATP9 antibody performance in complex tissue samples requires systematic approaches:

• Tissue preservation and fixation optimization:

  • Compare different fixatives (paraformaldehyde, glutaraldehyde, methanol) for optimal epitope preservation

  • Test variable fixation durations to balance structural preservation with epitope accessibility

  • For ATP9 in mitochondria, mild fixation conditions often preserve antigenicity better than harsh fixatives

• Antigen retrieval methods:

  • Systematically test heat-induced epitope retrieval at different pH values (citrate buffer pH 6.0, Tris-EDTA pH 9.0)

  • Evaluate enzymatic retrieval approaches (proteinase K, trypsin) at varying concentrations

  • Determine optimal retrieval duration to maximize signal without tissue degradation

• Signal amplification approaches:

  • Implement tyramide signal amplification for immunohistochemistry applications

  • Use biotin-streptavidin systems when standard detection methods yield insufficient signal

  • Consider polymer-based detection systems that reduce background while enhancing specific signal

• Background reduction strategies:

  • Pre-adsorb antibodies with tissue homogenates from ATP9-null models to remove non-specific binding

  • Test different blocking solutions (BSA, normal serum, commercial blockers) at various concentrations

  • Employ longer washing steps with gentle agitation to remove unbound antibody

• Multiplexing optimization:

  • When co-staining with other mitochondrial markers, carefully select antibodies raised in different host species

  • Determine optimal antibody sequence (primary antibodies applied sequentially vs. simultaneously)

  • Validate specificity of multiplexed signals using appropriate controls

• Tissue-specific protocol adjustments:

  • Modify permeabilization conditions based on tissue lipid content (brain vs. muscle)

  • Adjust antibody concentration and incubation time for tissues with varying ATP9 expression levels

  • Implement tissue-specific autofluorescence reduction steps (Sudan Black B for lipofuscin)

These optimization strategies should be systematically documented to establish reproducible protocols for different tissue types and experimental conditions.

How should researchers interpret contradictory results between ATP9 antibody signals and functional assays of ATP synthase activity?

When faced with discrepancies between ATP9 antibody signals and functional ATP synthase assays, researchers should consider these analytical approaches:

• Establish correlation thresholds: Not all changes in ATP9 levels directly correlate with ATP synthase activity. Researchers should determine the threshold of ATP9 expression necessary for minimal, optimal, and maximal ATP synthase function. Studies with nucleus-encoded ATP9 have shown that even significantly reduced ATP9 levels (compared to wild-type) can sustain respiratory growth, albeit at diminished rates (40-80% of wild-type oxygen consumption) .

• Evaluate post-translational modifications: ATP9 function may be regulated by modifications not detected by standard antibodies. Consider:

  • Using antibodies specific to phosphorylated, acetylated, or otherwise modified ATP9

  • Comparing results from antibodies targeting different epitopes of ATP9

  • Conducting mass spectrometry analysis to identify modifications affecting function

• Assess assembly status versus abundance: High ATP9 levels may not translate to high activity if assembly is impaired. Analyze:

  • ATP9 incorporation into complexes using blue native PAGE alongside Western blotting

  • The ratio of free versus assembled ATP9 using appropriate extraction conditions

  • The presence of assembly intermediates that may contain ATP9 but lack activity

• Examine stoichiometric relationships: ATP9 must be present in the correct stoichiometry relative to other subunits. Investigate:

  • The ratio of ATP9 to other key subunits (ATP6, ATP8)

  • Whether excess unassembled ATP9 might be detected by antibodies but not contributing to function

  • If altered stoichiometry affects enzyme kinetics rather than maximal activity

• Consider genetic background effects: In experimental systems with relocated ATP9 genes, genetic background may influence correlation between protein levels and function. For instance, studies showed that different expression systems (centromeric versus multicopy plasmids) yielded varying functional outcomes despite similar protein levels .

• Analyze temporal dynamics: Discrepancies may reflect different temporal sensitivities:

  • ATP9 may be detected before it's functionally incorporated

  • Activity may persist after ATP9 levels begin to decline

  • Turnover rates may differ between detection and activity assays

When interpreting such contradictory results, researchers should integrate multiple lines of evidence rather than relying solely on either antibody detection or functional assays.

What statistical approaches are most appropriate for quantifying ATP9 expression levels across different experimental conditions?

When quantifying ATP9 expression levels across experimental conditions, researchers should implement these statistical approaches:

These statistical approaches enhance the reliability and interpretability of ATP9 expression data, facilitating meaningful comparisons across experimental conditions.

How can researchers effectively distinguish between effects on ATP9 expression versus effects on ATP synthase assembly when interpreting antibody-based experiments?

Distinguishing between effects on ATP9 expression and ATP synthase assembly requires a multi-dimensional analytical approach:

• Complementary detection methods:

  • Compare ATP9 antibody signals in denaturing (SDS-PAGE) versus native (BN-PAGE) conditions

  • Use pulse-chase labeling with ATP9 antibody immunoprecipitation to track newly synthesized ATP9 and its assembly fate

  • Employ proximity ligation assays to detect interactions between ATP9 and other ATP synthase subunits in situ

• Differential extraction protocols:

  • Apply increasingly stringent detergent conditions to distinguish between free ATP9 and assembled complexes

  • Compare gentle extraction (digitonin) that preserves complexes versus harsh conditions (SDS) that solubilize total protein

  • Fractionate mitochondria to separate membrane-bound versus assembled ATP9 populations

• Genetic approaches for clarification:

  • Express ATP9 in backgrounds lacking other essential ATP synthase subunits to assess expression independent of assembly

  • Use inducible expression systems to modulate ATP9 levels and track assembly kinetics over time

  • Compare nucleus-encoded versus mitochondria-encoded ATP9 to identify expression versus assembly constraints

• Functional correlations:

  • Measure ATP9 levels alongside ATP synthesis rates to establish functional relevance

  • Assess proton conductance (a function requiring properly assembled ATP9 rings) versus total ATP9 protein

  • Evaluate oligomycin sensitivity as an indicator of proper ATP9 incorporation into functional complexes

• Structural analysis:

  • Use conformation-specific antibodies that recognize ATP9 only in certain assembly states

  • Combine antibody detection with structural techniques (electron microscopy, crosslinking mass spectrometry)

  • Apply super-resolution microscopy to visualize ATP9 distribution relative to assembled complexes

• Mathematical modeling:

  • Develop kinetic models incorporating both expression and assembly parameters

  • Use time-course data to distinguish rate-limiting steps in expression versus assembly

  • Implement sensitivity analysis to identify which processes most strongly influence experimental outcomes

These approaches allow researchers to deconvolute the complex relationship between ATP9 expression and its assembly into functional ATP synthase complexes, providing clearer interpretation of experimental results.

What strategies should researchers employ when developing ATP9 antibodies for studying its evolutionary relocation from mitochondrial to nuclear genomes?

Developing ATP9 antibodies for evolutionary studies requires specialized approaches:

• Epitope conservation analysis:

  • Perform multiple sequence alignment of ATP9 proteins from species representing different evolutionary stages of gene transfer

  • Identify conserved epitopes that remain unchanged regardless of encoding genome

  • Design antibodies targeting both conserved regions (for cross-species detection) and variable regions (for species-specific detection)

• Targeting relocation-specific modifications:

  • Develop antibodies recognizing mitochondrial targeting sequences present only in nuclear-encoded ATP9

  • Create antibodies specific to processing intermediates that occur during import of nuclear-encoded ATP9

  • Design antibodies against regions that undergo codon optimization during nuclear transfer

• Expression system considerations:

  • Validate antibodies in systems where both nuclear and mitochondrial versions co-exist

  • Test recognition in filamentous fungi like Podospora anserina where nuclear ATP9 genes naturally occur

  • Evaluate performance in experimental yeast systems where ATP9 has been artificially relocated

• Structural adaptation detection:

  • Create antibodies sensitive to conformational changes that accompany reduced hydrophobicity (a key adaptation enabling nuclear relocation)

  • Design epitopes that specifically recognize structural adaptations enabling import machinery recognition

  • Develop antibodies targeting regions involved in assembly that may be modified during genome relocation

• Experimental validation approaches:

  • Test antibody specificity against synthetic peptides representing ATP9 variants

  • Validate recognition using heterologous expression systems with ATP9 from different species

  • Compare performance in Δatp9 yeast complemented with nuclear ATP9 genes from evolutionarily diverse sources

This strategic approach enables researchers to create antibody tools specifically tailored for investigating the evolutionary journey of ATP9 from mitochondrial to nuclear genomes, illuminating a fundamental process in eukaryotic evolution.

How can researchers utilize ATP9 antibodies in combination with other techniques to study mitochondrial disease pathogenesis?

Integrating ATP9 antibodies with complementary techniques creates powerful approaches for mitochondrial disease research:

• Combined proteomics and immunodetection workflow:

  • Use ATP9 antibodies for immunoprecipitation followed by mass spectrometry

  • Identify ATP9 interaction partners that may be altered in disease states

  • Validate mass spectrometry findings with targeted co-immunoprecipitation experiments

• Multi-omics integration strategy:

  • Correlate ATP9 antibody signals with transcriptomic data to identify expression-translation discordance

  • Combine with metabolomics to link ATP9 defects to specific metabolic signatures

  • Integrate with genomic data to connect genetic variants with ATP9 expression/assembly phenotypes

• Live-cell imaging approaches:

  • Use ATP9 antibody fragments conjugated to quantum dots for single-molecule tracking

  • Combine with mitochondrial potential indicators to correlate ATP9 localization with function

  • Implement FRET sensors to detect ATP9 conformational changes in living cells

• Patient-derived model systems:

  • Apply ATP9 antibodies to patient-derived fibroblasts, iPSCs, and differentiated tissues

  • Compare ATP9 expression, localization, and assembly between patient and control samples

  • Validate findings across multiple patient lines to establish disease-specific patterns

• CRISPR-based functional genomics:

  • Use ATP9 antibodies to assess the impact of CRISPR-induced mutations in ATP9 interactors

  • Create cellular models with tagged endogenous ATP9 for dynamic studies

  • Implement CRISPR activation/interference systems to modulate ATP9 expression

• Therapeutic monitoring platform:

  • Develop high-throughput immunoassays using ATP9 antibodies for drug screening

  • Track ATP9 assembly in response to candidate therapeutics

  • Establish biomarker panels including ATP9 measurements for clinical trials

This integrated approach leverages ATP9 antibodies as part of a comprehensive toolkit for understanding mitochondrial disease mechanisms, potentially leading to novel diagnostic and therapeutic strategies.

What are the optimal conditions for ATP9 antibody storage and handling to maintain long-term activity?

Ensuring optimal ATP9 antibody performance requires careful attention to storage and handling conditions:

For ATP9 antibodies specifically, researchers should:

• Test performance periodically against positive controls (wild-type mitochondria) and negative controls (Δatp9 samples)

• Consider the recognized epitope when choosing storage conditions, as hydrophobic epitopes may require special stabilization

• Document batch-to-batch variation when using different lots of the same antibody

These practices ensure consistent and reliable results when using ATP9 antibodies across extended research projects.

What considerations should researchers take into account when designing experiments to detect post-translational modifications of ATP9?

Detecting post-translational modifications (PTMs) of ATP9 requires specialized experimental design:

• Sample preparation optimization:

  • Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate) for phosphorylation studies

  • Add deacetylase inhibitors (trichostatin A, nicotinamide) when investigating acetylation

  • Use fresh samples whenever possible, as PTMs can be lost during storage

  • Optimize lysis conditions to preserve PTMs while effectively solubilizing membrane-embedded ATP9

• PTM-specific antibody selection:

  • When available, use antibodies specifically recognizing modified ATP9 (phospho-ATP9, acetyl-ATP9)

  • For novel PTMs, consider developing custom antibodies against modified peptides

  • Validate PTM antibodies using samples treated with modifying or demodifying enzymes

• Enrichment strategies:

  • Implement immunoprecipitation with ATP9 antibodies followed by PTM-specific detection

  • Use immobilized metal affinity chromatography (IMAC) to enrich phosphorylated ATP9

  • Apply PTM-specific enrichment methods before mass spectrometry analysis

• Confirmation approaches:

  • Treat samples with specific enzymes (phosphatases, deacetylases) to confirm PTM identity

  • Use site-directed mutagenesis to convert modifiable residues to non-modifiable ones

  • Compare PTM patterns between wild-type and ATP9 relocated to the nucleus

• Detection methods combination:

  • Complement antibody detection with mass spectrometry for unbiased PTM mapping

  • Use Phos-tag™ SDS-PAGE to separate phosphorylated from non-phosphorylated forms

  • Apply 2D gel electrophoresis to separate ATP9 variants with different modification patterns

• Functional correlation analysis:

  • Design experiments that link detected PTMs to ATP synthase assembly or activity

  • Investigate whether PTMs differ between free ATP9 and that incorporated into complexes

  • Examine whether nucleus-encoded ATP9 displays different modification patterns than mitochondria-encoded versions

These considerations enable researchers to accurately detect and characterize post-translational modifications of ATP9, providing insights into regulatory mechanisms affecting mitochondrial function.