ATP6V1E1 Antibody

What is ATP6V1E1 and what is its role in cellular function?

ATP6V1E1 is a subunit of the peripheral V1 complex of vacuolar ATPase (V-ATPase), a multisubunit enzyme composed of a peripheral complex (V1) that hydrolyzes ATP and a membrane integral complex (V0) that translocates protons. In humans, the canonical protein has 81 amino acid residues with a calculated molecular mass of 9.4 kDa (though observed at ~31 kDa in experimental conditions). ATP6V1E1 is essential for the assembly and catalytic function of V-ATPase, which is responsible for acidifying various intracellular compartments in eukaryotic cells and, in some cell types, the extracellular environment .

The protein belongs to the V-ATPase e1/e2 subunit family and is ubiquitously expressed across many tissue types, with subcellular localization primarily in membranes. It undergoes post-translational modifications, including glycosylation .

What are the common synonyms and orthologs for ATP6V1E1?

Understanding the various nomenclature is essential for comprehensive literature searches:

The protein has been identified across multiple species, allowing for comparative studies .

How do I select the appropriate ATP6V1E1 antibody for my research?

When selecting an ATP6V1E1 antibody, consider these critical factors:

• Application compatibility: Different antibodies are optimized for specific applications. From the search results, Western Blot, ELISA, and Immunohistochemistry are the most common applications for ATP6V1E1 antibodies .

• Species reactivity: Ensure the antibody recognizes your species of interest. Available antibodies show reactivity with human, mouse, and rat samples, with some also recognizing other species .

• Clonality: Choose between:

  • Polyclonal antibodies: Broader epitope recognition but potential batch variation

  • Monoclonal antibodies: Consistent specificity but potentially narrower epitope recognition

  • Recombinant monoclonal antibodies: Offer consistency and reproducibility

• Immunogen region: Consider whether the antibody targets a region of interest within the protein structure, particularly if studying specific domains or variants .

• Validation data: Examine the validation data provided by manufacturers, including Western blot images, IHC staining patterns, and specificity testing .

What are the optimal conditions for using ATP6V1E1 antibodies in Western blotting?

For optimal Western blotting with ATP6V1E1 antibodies, follow these evidence-based recommendations:

• Sample preparation: Total protein extraction with Laemmli Sample Buffer has been successfully used for ATP6V1E1 Western blots .

• Expected molecular weight: While the calculated molecular weight is ~26 kDa, ATP6V1E1 is typically observed at approximately 31 kDa on SDS-PAGE gels .

• Recommended dilutions: Based on validated protocols:

  • Polyclonal antibodies: 1:1000-1:6000

  • Monoclonal antibodies: 1:1000-1:4000

  • For specific antibodies like ab111733: 1:3000

• Blocking conditions: 5% non-fat dry milk in TBST has been successfully used .

• Detection methods: Horseradish-peroxidase-conjugated secondary antibodies at approximately 1:1500 dilution have shown good results .

• Positive controls: HeLa cells, human liver tissue, human/mouse brain tissue, and HEK-293 cells have been validated as positive controls for Western blotting .

How can I optimize immunohistochemistry protocols for ATP6V1E1 detection?

For effective immunohistochemical (IHC) detection of ATP6V1E1:

• Antigen retrieval: Two validated methods are:

  • TE buffer pH 9.0 (preferred method)

  • Citrate buffer pH 6.0 (alternative method)

• Optimal dilutions: Typically 1:50-1:500 for most ATP6V1E1 antibodies, with specific recommendations varying by product .

• Validated tissue samples:

  • Mouse tissues: testis, kidney

  • Human tissues: testis

  • Rat tissues: kidney

• Detection systems: Both ABC Detection Kits and polymer-based systems have been used successfully for ATP6V1E1 IHC .

• Controls: Include positive tissue controls (based on known expression patterns) and negative controls (omitting primary antibody) to validate staining specificity.

What methodological considerations are important for immunoprecipitation experiments with ATP6V1E1 antibodies?

When performing immunoprecipitation (IP) with ATP6V1E1 antibodies:

• Antibody amount: Use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate .

• Validated tissue samples: Mouse brain tissue has been successfully used for ATP6V1E1 IP experiments .

• Experimental controls: When studying protein interactions, consider these controls used in published V-ATPase interactome studies:

  • Beads-only controls (no antibody)

  • IPs from knockout models lacking the target protein

  • Peptide competition controls (antibodies pre-incubated with immunizing peptide)

• Interaction validation: Following IP-MS experiments, validate interactions by Western blotting when functional antibodies are available for the interacting proteins .

• Technical considerations: Cross-linking of antibodies to beads may help reduce background and improve specificity, particularly when studying protein complexes like V-ATPase .

How can ATP6V1E1 antibodies be used to study V-ATPase complex assembly and function?

ATP6V1E1 antibodies can provide insights into V-ATPase complex assembly and function through several approaches:

• Co-immunoprecipitation studies: ATP6V1E1 antibodies have been used to identify protein-protein interactions within the V-ATPase complex and with novel interacting partners. Research has revealed interactions with other V-ATPase subunits and proteins like SLC9A3R1 (NHERF1) and SLC9A3R2 (NHERF2) .

• Structural analysis: Antibodies can be used to validate structural models generated through homology modeling. Previous research has used the Saccharomyces cerevisiae V-ATPase structure (PDB: 3J9T) as a template for modeling human V-ATPase components .

• Subcellular localization: Immunofluorescence using ATP6V1E1 antibodies can reveal the distribution of V-ATPase complexes across different cellular compartments and determine whether mutations affect localization patterns .

• Functional studies: Combining ATP6V1E1 immunodetection with functional assays (such as lysosomal acidification or protein trafficking) can correlate V-ATPase complex assembly with functional outcomes .

• Mutational analysis: ATP6V1E1 antibodies have been essential in studying how mutations affect protein expression, stability, and complex assembly, particularly in contexts of disease-causing variants .

What are the methodological approaches for studying ATP6V1E1 in disease models?

When investigating ATP6V1E1 in disease models, consider these methodological approaches:

• Patient-derived samples: ATP6V1E1 antibodies have been used to detect protein expression in patient-derived cells, particularly in cases of mutations causing autosomal-recessive disorders .

• Knockout/knockdown validation:

  • siRNA or CRISPR-based approaches targeting ATP6V1E1 provide important controls for antibody specificity

  • Patient samples with ATP6V1E1 mutations serve as natural models for studying protein function

• Functional readouts:

  • Golgi trafficking: Assessing GOLPH4 localization following brefeldin A treatment

  • Lysosomal function: Monitoring degradative capacity and pH

  • ECM homeostasis: Evaluating extracellular matrix components like ICAM-1

• Comparative analysis: Comparing ATP6V1E1 with its closely related family member ATP6V1A, which has also been implicated in disease, can provide insights into shared and distinct functions .

• Structural impact assessment: Using antibodies to validate expression of mutant proteins, combined with computational modeling to predict structural impacts of disease-associated mutations .

How can ATP6V1E1 antibodies be used in multiplex imaging and high-throughput analytical approaches?

For advanced multiplex imaging and high-throughput analyses:

• Conjugation-ready formats: Several ATP6V1E1 antibodies are available in conjugation-ready formats designed for:

  • Fluorochrome labeling

  • Metal isotope tagging (for mass cytometry)

  • Oligonucleotide conjugation

  • Enzymatic labeling

• Matched antibody pairs: For quantitative analyses like cytometric bead arrays or ELISAs, validated matched pairs are available:

  • Example: MP00628-1 uses 83636-1-PBS as capture and 83636-4-PBS as detection antibodies

• Multiplex imaging applications: BSA and azide-free formulations enable custom labeling for multiplexed imaging techniques that allow simultaneous detection of ATP6V1E1 alongside other proteins of interest .

• Flow cytometry: Some ATP6V1E1 antibodies have been validated for intracellular flow cytometry, allowing high-throughput analysis of expression levels across cell populations .

• Technical considerations: For multiplex applications, recombinant antibodies may offer advantages due to their batch-to-batch consistency and defined specificity .

What are common issues when using ATP6V1E1 antibodies and how can they be resolved?

When working with ATP6V1E1 antibodies, researchers may encounter these challenges:

• Unexpected molecular weight: While the calculated molecular weight is ~26 kDa, ATP6V1E1 is typically observed at ~31 kDa in experimental conditions. This discrepancy could be due to post-translational modifications such as glycosylation .

  Solution: Include positive controls with known ATP6V1E1 expression to confirm the appropriate band size.

• Cross-reactivity concerns: ATP6V1E1 belongs to a family of related proteins, including ATP6V1E2, which could lead to cross-reactivity.

  Solution: Validate specificity using:

  • Knockout/knockdown samples

  • Peptide competition assays

  • Testing in multiple applications

• Variable staining patterns: Since ATP6V1E1 is ubiquitously expressed but with varying levels across tissues, inconsistent staining might occur.

  Solution: Optimize antibody concentration for each tissue type and include appropriate positive and negative controls.

• Storage-related issues: Antibody performance can degrade with improper storage.

  Solution: Follow manufacturer recommendations for storage conditions. Most ATP6V1E1 antibodies should be stored at -20°C, with aliquoting recommended to avoid repeated freeze-thaw cycles .

How can I validate the specificity of ATP6V1E1 antibodies in my experimental system?

To ensure ATP6V1E1 antibody specificity:

• Genetic validation approaches:

  • siRNA knockdown of ATP6V1E1

  • CRISPR knockout models

  • Patient-derived cells with ATP6V1E1 mutations

• Peptide competition: Pre-incubating the antibody with the immunizing peptide should abolish specific signals. This approach has been used successfully in V-ATPase interaction studies .

• Orthogonal detection methods: Compare results using:

  • Multiple antibodies targeting different epitopes

  • Mass spectrometry validation of immunoprecipitated proteins

  • RNA expression correlation with protein detection

• Enhanced validation techniques: Some commercial antibodies undergo enhanced validation through:

  • Orthogonal RNAseq validation

  • Independent antibody verification

  • Multiple application testing

• Multiple detection methods: Confirm findings across different methods (Western blot, IHC, IF) to ensure consistent results across applications.

How are ATP6V1E1 antibodies being used to investigate novel disease associations?

ATP6V1E1 antibodies are instrumental in uncovering new disease associations:

• Autosomal-recessive disorders: Mutations in ATP6V1E1 have been identified as causes of distinct metabolic and multisystemic cutis laxa entities. Antibodies have helped demonstrate how these mutations affect:

  • V-ATPase structure and assembly

  • Protein glycosylation

  • Golgi trafficking

  • Lysosomal function

  • ECM homeostasis and architecture

• Infection pathways: ATP6V1E1 plays a role in phagosome acidification, with research showing that mycobacterial protein tyrosine phosphatase PtpA can interact with this pathway, potentially contributing to pathogenesis .

• Cancer research: The Human Protein Atlas project has used ATP6V1E1 antibodies to map expression across both normal and cancer tissues, providing insights into potential roles in malignancy .

• Cellular homeostasis: ATP6V1E1 antibodies have revealed the protein's importance in protein degradation, receptor-mediated endocytosis, and neurotransmitter uptake, suggesting potential links to neurodegenerative disorders .

What are the latest methodological advances in ATP6V1E1 antibody development and application?

Recent advances in ATP6V1E1 antibody technology include:

• Recombinant antibody production: In-house recombinant technology enables unrivalled batch-to-batch consistency, easy scale-up, and future security of supply for ATP6V1E1 antibodies .

• Conjugation-ready formats: Specialized formulations without BSA and azide facilitate direct conjugation for applications requiring labeled antibodies .

• Validated matched pairs: Development of validated antibody pairs for sandwich assays enables quantitative detection of ATP6V1E1 in complex biological samples .

• Enhanced validation approaches: Antibody manufacturers are implementing rigorous validation workflows including:

  • Orthogonal RNAseq validation

  • Testing across multiple applications

  • Extensive species cross-reactivity testing

• Human Protein Atlas integration: ATP6V1E1 antibodies have been incorporated into systematic protein mapping efforts, providing standardized validation data across hundreds of tissues and subcellular localization patterns .

What considerations are important when designing experiments to study ATP6V1E1 variants and mutations?

When investigating ATP6V1E1 variants and mutations:

• Appropriate controls: For disease-associated variants, include:

  • Wild-type ATP6V1E1 controls

  • Other known disease-causing mutations for comparison

  • Benign polymorphisms to distinguish pathogenic from non-pathogenic changes

• Structural analysis integration: Combine antibody-based detection with structural modeling approaches:

  • Use homology models based on S. cerevisiae V-ATPase structure

  • Analyze potential disruptions to protein-protein interactions

  • Assess hydrogen bonding and structural integrity changes

• Functional readouts: Beyond expression levels, assess:

  • Protein stability and turnover rates

  • Complex assembly efficiency

  • Subcellular localization patterns

  • Functional outcomes (acidification, trafficking)

• Interaction studies: Determine if mutations affect known protein-protein interactions:

  • With other V-ATPase subunits

  • With regulatory partners like SLC9A3R1/2

  • With novel interactors identified through IP-MS approaches

• Species considerations: Due to high conservation, studies in model organisms can provide insights, but species-specific differences should be considered when translating findings to human disease .