ATP6V1A Antibody

What is the biological function of ATP6V1A protein?

ATP6V1A forms the A subunit of the V-ATPase complex, which is essential for maintaining the correct pH of endosomes, lysosomes, and other intracellular compartments . The V-ATPase is a multisubunit enzyme that mediates acidification of eukaryotic intracellular organelles, a process necessary for protein sorting, zymogen activation, receptor-mediated endocytosis, and synaptic vesicle proton gradient generation . The V-ATPase has two main domains: a cytosolic V1 domain containing the ATP catalytic site, and a transmembrane V0 domain . ATP6V1A specifically encodes one of two V1 domain A subunit isoforms and is found in all tissues .

What sample types have been validated for ATP6V1A antibody applications?

ATP6V1A antibodies have been validated for detection in multiple human and mouse samples. Specifically, positive Western blot detection has been confirmed in:

These validations have been performed using different antibody clones, including monoclonal and polyclonal formulations .

What are the recommended applications for ATP6V1A antibodies?

ATP6V1A antibodies have been validated for several research applications with specific recommended dilutions:

Researchers should note that optimal dilutions may be sample-dependent and should be determined empirically for each experimental system .

How can ATP6V1A antibodies be used to investigate V-ATPase complex assembly?

For investigating V-ATPase complex assembly, immunoprecipitation (IP) followed by mass spectrometry or co-immunoprecipitation (co-IP) with antibodies against other V-ATPase subunits provides comprehensive insights. The experimental approach should involve:

• Cell lysis under native conditions using mild detergents (e.g., 1% NP-40 or 0.5% Triton X-100) to preserve protein-protein interactions

• IP with ATP6V1A antibody conjugated to magnetic or agarose beads

• Western blot analysis with antibodies against other V-ATPase subunits or mass spectrometry to identify interaction partners

• Comparison between normal and pathological samples to detect assembly defects

This approach can detect association defects between ATP6V1A and other V-ATPase components, particularly in conditions like cutis laxa syndrome where ATP6V1A mutations affect complex formation and function .

What experimental controls are essential when studying ATP6V1A expression in cancer tissues?

When investigating ATP6V1A expression in cancer tissues, particularly gastric cancer where expression is significantly elevated, several critical controls must be implemented:

• Positive tissue controls: Include kidney and brain tissues which naturally express ATP6V1A at detectable levels

• Antibody validation: Perform peptide competition assays to confirm specificity

• Expression normalization: Use multiple housekeeping genes for qRT-PCR studies, as some traditional housekeeping genes may vary in cancer tissues

• Sample stratification: Since ATP6V1A expression correlates with prognosis in gastric cancer, samples should be stratified by clinical parameters (stage, grade, treatment history)

• Parallel YY1 expression analysis: Given that YY1 transcriptionally regulates ATP6V1A, concurrent analysis of YY1 provides important mechanistic context

These controls help distinguish between causative and consequential changes in ATP6V1A expression in cancer progression .

How should researchers troubleshoot non-specific binding when using ATP6V1A antibodies?

Non-specific binding is a common issue when working with ATP6V1A antibodies. A systematic troubleshooting approach should include:

• Antibody titration: Test dilutions beyond the recommended range (e.g., 1:250-1:2000 for WB) to identify optimal signal-to-noise ratio

• Blocking optimization: Compare different blocking agents (BSA, non-fat milk, commercial blockers) at varying concentrations (3-5%)

• Wash stringency adjustment: Increase TBST concentration (0.1% to 0.3% Tween-20) or number of wash steps

• Sample preparation modification: Ensure complete protein denaturation for WB (boil samples with SDS and DTT/β-mercaptoethanol)

• Knockout/knockdown validation: Include ATP6V1A knockout/knockdown samples as negative controls when available

• Cross-reactivity testing: Test antibody specificity against recombinant ATP6V1A2 (the other V1 domain A subunit isoform) to ensure isoform specificity

If non-specific bands persist around 70 kDa, consider that they may represent alternatively spliced variants, as ATP6V1A has transcript variants derived from alternative polyadenylation .

How should researchers interpret ATP6V1A expression changes in gastric cancer studies?

Interpreting ATP6V1A expression changes in gastric cancer requires careful consideration of several factors:

These considerations highlight the complexity of ATP6V1A's role in cancer biology and suggest it may serve as a potential prognostic biomarker rather than a straightforward oncogenic driver .

What factors affect the reproducibility of ATP6V1A immunodetection across different experimental systems?

Several critical factors influence the reproducibility of ATP6V1A detection:

• Antibody clone selection: Different clones (e.g., AB01/4F4 monoclonal vs. polyclonal antibodies) may recognize different epitopes, affecting sensitivity and specificity

• Sample preparation methods:

  • For Western blotting: Protein extraction buffers (RIPA vs. NP-40) can affect epitope accessibility

  • For IHC: Antigen retrieval methods significantly impact detection (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Expression level variations: ATP6V1A expression varies by tissue type, with different baseline expression levels in kidney versus brain or testis tissues

• Post-translational modifications: These may affect epitope recognition and apparent molecular weight

• Cross-reactivity with related proteins: The V-ATPase complex contains multiple subunits with structural similarities

• Fixation artifacts: For IHC/IF applications, fixation duration and conditions can create artifacts or mask epitopes

Researchers should validate each new antibody lot in their specific experimental system and include appropriate positive controls (e.g., HeLa cells for Western blotting) .

How can researchers distinguish between ATP6V1A detection and other V-ATPase subunits?

Distinguishing ATP6V1A from other V-ATPase subunits requires a multi-faceted approach:

• Molecular weight discrimination: ATP6V1A has a distinctive 68-72 kDa band, differentiating it from other V-ATPase subunits which range from approximately 16 kDa (c subunits) to 100 kDa (a subunits)

• Epitope selection validation: Choose antibodies raised against unique regions of ATP6V1A not conserved in other subunits. For example, antibodies generated against amino acids 1-350 of human ATP6V1A target regions distinct from other V-ATPase components

• Isoform-specific detection: Ensure the antibody specifically recognizes ATP6V1A rather than the related ATP6V1A2 isoform by checking the immunogen sequence

• siRNA/shRNA verification: Validate antibody specificity through ATP6V1A knockdown experiments, confirming signal reduction

• Parallel detection strategy: Use multiple antibodies targeting different epitopes of ATP6V1A to confirm specificity

• Mass spectrometry confirmation: For critical experiments, immunoprecipitation followed by mass spectrometry can definitively identify the detected protein

These approaches collectively increase confidence in the specific detection of ATP6V1A rather than related V-ATPase components or other cross-reactive proteins.

How can ATP6V1A antibodies be used to study cutis laxa syndrome and other genetic disorders?

ATP6V1A antibodies serve as valuable tools for investigating cutis laxa syndrome and related genetic disorders through several methodological approaches:

• Mutation impact analysis: Compare ATP6V1A protein expression, localization, and stability between patient-derived cells and controls using Western blotting and immunofluorescence to determine how specific mutations affect protein levels and cellular distribution

• V-ATPase complex integrity assessment: Employ co-immunoprecipitation with ATP6V1A antibodies followed by detection of other V-ATPase subunits to determine if mutations disrupt complex assembly

• Functional acidification assays: Combine ATP6V1A immunostaining with LysoTracker or pH-sensitive fluorescent proteins to correlate ATP6V1A localization with organellar pH regulation defects

• Patient tissue immunohistochemistry: Apply validated ATP6V1A antibodies (1:20-1:200 dilution) to patient tissue sections with appropriate antigen retrieval (TE buffer pH 9.0) to assess protein expression patterns in affected tissues

• Model system validation: Use ATP6V1A antibodies to confirm knockdown/knockout efficiency or mutation introduction in disease models (cell lines, organoids, animal models)

These approaches help elucidate how inherited ATP6V1A mutations lead to the neurological and cardiopulmonary complications characteristic of cutis laxa syndrome .

What methodological approaches can detect changes in ATP6V1A localization during cancer progression?

Detecting changes in ATP6V1A localization during cancer progression requires specialized methodological approaches:

• Subcellular fractionation with immunoblotting:

  • Separate cellular components (membrane, cytosol, organelles) through differential centrifugation

  • Perform Western blotting with ATP6V1A antibodies (1:500-1:1000 dilution)

  • Use compartment-specific markers (Na⁺/K⁺-ATPase for plasma membrane, LAMP1 for lysosomes) to confirm fraction purity

• Confocal immunofluorescence microscopy:

  • Apply ATP6V1A antibodies to fixed cancer cell lines or tissue sections

  • Co-stain with markers for specific compartments (early endosomes, late endosomes, lysosomes, plasma membrane)

  • Perform z-stack imaging to create 3D reconstructions of protein localization

  • Quantify colocalization coefficients (Pearson's or Mander's) to measure association with compartment markers

• Tissue microarray analysis:

  • Apply immunohistochemistry protocols (1:20-1:200 antibody dilution) to tumor tissue microarrays representing different cancer stages

  • Score not only expression intensity but also subcellular distribution patterns

  • Correlate localization patterns with clinical outcomes

• Live-cell imaging approaches:

  • Generate ATP6V1A-GFP fusion constructs to track localization in living cells

  • Validate fusion protein localization matches endogenous protein using ATP6V1A antibodies

  • Monitor localization changes during stimulated migration or invasion

These approaches can reveal whether ATP6V1A relocalization to specific compartments (e.g., plasma membrane vs. lysosomes) correlates with the better prognosis observed in some gastric cancer patients with higher ATP6V1A expression .

How can ATP6V1A antibodies be integrated with proteomics approaches to study V-ATPase complex dynamics?

Integration of ATP6V1A antibodies with advanced proteomics offers powerful insights into V-ATPase complex assembly, regulation, and dynamics:

• Proximity-dependent biotinylation (BioID/TurboID):

  • Generate ATP6V1A fusion with biotin ligase

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

  • Validate interactions using co-immunoprecipitation with ATP6V1A antibodies

  • This approach captures transient interactions and spatial relationships within the V-ATPase complex

• Crosslinking mass spectrometry (XL-MS):

  • Perform chemical crosslinking of intact V-ATPase complexes

  • Immunoprecipitate using ATP6V1A antibodies

  • Analyze crosslinked peptides by mass spectrometry

  • Map interaction interfaces between ATP6V1A and other subunits

• Pulse-chase proteomics:

  • Label newly synthesized proteins with amino acid isotopes

  • Immunoprecipitate ATP6V1A at different time points

  • Analyze assembly kinetics of the V-ATPase complex

  • Identify factors affecting complex stability and turnover

• Quantitative interactomics in disease models:

  • Compare ATP6V1A interaction partners between normal and disease states

  • Apply antibodies for immunoprecipitation followed by SILAC or TMT-based quantitative proteomics

  • Identify altered regulatory interactions in conditions like cancer or cutis laxa syndrome

These integrative approaches provide dynamic information beyond static protein levels, revealing how ATP6V1A contributes to functional V-ATPase assembly in health and disease.

What considerations are important when designing ATP6V1A knockdown validation experiments?

When designing ATP6V1A knockdown experiments to validate antibody specificity or study functional effects, several critical considerations must be addressed:

• Knockdown approach selection:

  • siRNA: Best for transient knockdown and rapid validation

  • shRNA: Preferred for stable knockdown in long-term experiments

  • CRISPR/Cas9: For complete knockout studies, noting that complete ATP6V1A deletion may affect cell viability

• Target sequence design:

  • Avoid regions with similarity to ATP6V1A2 (the other V1 domain A isoform)

  • Design multiple targeting sequences to control for off-target effects

  • Include non-targeting control sequences with similar GC content

• Transfection/transduction optimization:

  • Determine optimal conditions for each cell type (reagent, cell density, incubation time)

  • Establish knockdown time course to identify peak effect window

  • Consider co-transfection of fluorescent markers to identify transfected cells

• Validation methodology:

  • Confirm knockdown at mRNA level by qRT-PCR

  • Verify protein reduction by Western blot with ATP6V1A antibodies (1:500-1:1000 dilution)

  • Assess functional consequences through organelle acidification assays

• YY1 knockdown as complementary approach:

  • Given YY1's role in regulating ATP6V1A expression, parallel YY1 knockdown provides mechanistic insight

  • Compare direct ATP6V1A knockdown with indirect reduction via YY1 knockdown

• Rescue experiments:

  • Reintroduce siRNA/shRNA-resistant ATP6V1A constructs to confirm phenotype specificity

  • Include ATP6V1A mutants mimicking disease variants to assess functional consequences

These design considerations ensure robust and specific ATP6V1A knockdown validation, critical for antibody specificity confirmation and functional studies.

What are the key considerations for selecting the optimal ATP6V1A antibody for specific research applications?

Selecting the optimal ATP6V1A antibody requires careful evaluation of multiple factors based on the intended application:

• Clone type consideration:

  • Monoclonal antibodies (e.g., AB01/4F4) offer high specificity and batch consistency ideal for standardized assays

  • Polyclonal antibodies provide higher sensitivity through multiple epitope recognition, beneficial for detection of low-abundance proteins

• Application-specific validation:

  • For Western blotting: Confirm single band detection at the expected 68-72 kDa size

  • For IHC/IF: Verify specific cellular/tissue distribution patterns matching known biology

  • For IP: Validate efficient pull-down of the target protein

  • For multiplexing: Test for compatibility with other antibodies in co-staining protocols

• Species reactivity requirements:

  • Confirm validation in relevant species (human, mouse, etc.)

  • Consider cross-reactivity when working with model organisms

• Epitope considerations:

  • N-terminal vs. C-terminal targeting may affect detection of splice variants

  • Ensure epitope accessibility in the experimental conditions (native vs. denatured)

  • Evaluate potential post-translational modification interference with epitope recognition

• Experimental controls integration:

  • Plan for appropriate positive controls (HeLa cells, kidney tissue)

  • Include knockout/knockdown validation when possible

By systematically evaluating these factors, researchers can select ATP6V1A antibodies optimally suited to their specific experimental questions and methodologies.

How might ATP6V1A antibodies contribute to understanding the paradoxical role of this protein in cancer progression?

ATP6V1A antibodies are instrumental in elucidating the complex and seemingly paradoxical role of ATP6V1A in cancer, particularly the observation that while expression is elevated in gastric cancers, higher levels correlate with better prognosis . Several research directions can leverage these antibodies:

• Comprehensive expression profiling:

  • Apply ATP6V1A antibodies to tissue microarrays across cancer types and stages

  • Correlate expression patterns with clinical outcomes

  • Integrate with genomic data to identify patient subgroups with different prognostic implications

• Functional complex assembly analysis:

  • Investigate whether ATP6V1A overexpression leads to functional or dysfunctional V-ATPase complexes

  • Use co-immunoprecipitation with ATP6V1A antibodies followed by detection of other V-ATPase subunits

  • Determine if the stoichiometry of complex components affects functionality and cancer progression

• Microenvironmental pH regulation studies:

  • Combine ATP6V1A immunostaining with pH-sensitive probes

  • Investigate how ATP6V1A expression affects intracellular and extracellular pH

  • Correlate pH regulation with invasive and metastatic potential

• YY1-ATP6V1A axis investigation:

  • Study how the transcription factor YY1 regulates ATP6V1A in different cancer contexts

  • Use ChIP assays with YY1 antibodies to confirm binding to the ATP6V1A promoter

  • Investigate potential feedback mechanisms between V-ATPase activity and YY1 expression

• Therapeutic response correlation:

  • Assess whether ATP6V1A expression levels predict response to specific cancer treatments

  • Investigate if V-ATPase inhibitors show different efficacy based on ATP6V1A expression patterns

These approaches utilizing ATP6V1A antibodies may reveal how this protein can be both upregulated in cancer yet associated with better outcomes, potentially identifying new prognostic markers or therapeutic targets.