ATP6V1E2 Antibody

What is ATP6V1E2 and why is it an important research target?

ATP6V1E2 (ATPase, H+ transporting, lysosomal 31kDa, V1 subunit E2) is a protein component of vacuolar ATPase complexes responsible for acidification of intracellular compartments. The protein functions as part of the V1 domain of V-ATPase, which is essential for the catalytic ATP hydrolysis that drives proton transport. Researching ATP6V1E2 is valuable because V-ATPases play critical roles in numerous cellular processes including membrane trafficking, protein degradation, neurotransmitter uptake, and maintenance of pH homeostasis. ATP6V1E2's specific 226 amino acid sequence translates to a calculated molecular weight of 26 kDa, though it typically appears as a 31-33 kDa band in experimental conditions due to post-translational modifications .

How does ATP6V1E2 antibody reactivity differ across species models?

The commercially available ATP6V1E2 antibodies predominantly demonstrate cross-reactivity with human, mouse, and rat samples as confirmed by multiple manufacturers . Certain antibody clones may show expanded reactivity profiles that include bovine, canine, and ovine samples, though validation intensity varies across these additional species . When designing cross-species experiments, researchers should verify the specific epitope recognition patterns, as antibodies targeting different amino acid sequences (such as AA 1-226 versus AA 71-120) may exhibit different cross-reactivity profiles . For comparative studies between mammalian models, prioritize antibodies with documented validation across all relevant species or conduct preliminary validation tests to confirm consistent epitope recognition before proceeding with critical experiments.

What are the optimal storage conditions for maintaining ATP6V1E2 antibody integrity over extended research projects?

To maintain maximum reactivity and specificity throughout longitudinal research projects, ATP6V1E2 antibodies should be stored at -20°C in their shipped buffer solutions, which typically contain PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, most commercial preparations remain stable for approximately one year after shipment . For antibodies at working concentrations (0.05-0.1 mg/ml), avoiding repeated freeze-thaw cycles is crucial as this significantly decreases antibody performance . Rather than repeatedly thawing the stock solution, prepare small aliquots upon initial receipt. For the 20μl size preparations that contain 0.1% BSA, aliquoting is not necessary for -20°C storage . When designing experimental timelines, factor in possible performance variations when using antibodies approaching their one-year stability threshold.

What are the validated applications and optimal working dilutions for ATP6V1E2 antibody in different experimental contexts?

ATP6V1E2 antibodies have been successfully validated across multiple experimental applications, each requiring specific optimization:

For Western blotting, start with a 1:500 dilution and adjust based on signal intensity . For immunoprecipitation, the antibody amount should be scaled proportionally to total protein concentration, beginning with 0.5 μg antibody per mg of lysate for initial optimization . Each experimental system requires independent titration to achieve optimal signal-to-noise ratios, particularly when working with different tissue or cell types than those listed in validation data .

What protein extraction and sample preparation protocols are optimal for detecting ATP6V1E2 in different tissue and cell types?

For efficient ATP6V1E2 detection, extraction protocols should account for the protein's membrane association and subcellular localization. For neural tissues (where ATP6V1E2 has been well validated), use extraction buffers containing mild detergents (0.5-1% Triton X-100 or NP-40) supplemented with protease inhibitors . For cultured cells such as MCF-7, a RIPA buffer system provides effective extraction while maintaining protein integrity .

Sample preparation should include:

• Tissue homogenization in cold buffer (4°C) using mechanical disruption

• Incubation with extraction buffer for 30 minutes on ice with periodic vortexing

• Centrifugation at 12,000×g for 15 minutes at 4°C

• Collection of supernatant and protein quantification

• Sample denaturation at 95°C for 5 minutes in reducing SDS-PAGE loading buffer

For immunoprecipitation applications, gentler lysis conditions may better preserve protein-protein interactions that could be biologically relevant to ATP6V1E2 function. When analyzing tissues not previously validated (beyond brain tissue and MCF-7 cells), preliminary experiments comparing different extraction methods are advisable to establish optimal detection conditions .

How should researchers design proper controls when using ATP6V1E2 antibodies in their experiments?

A comprehensive control strategy for ATP6V1E2 antibody experiments should include:

• Positive Controls: Include mouse brain tissue lysate or MCF-7 cell lysate as validated positive controls that consistently show 31-33 kDa bands .

• Negative Controls:

  • Primary antibody omission (for non-specific secondary antibody binding)

  • Isotype control (rabbit IgG at equivalent concentration)

  • Non-expressing or low-expressing tissue/cell types

• Specificity Controls:

  • Pre-absorption with immunizing peptide (if available)

  • siRNA knockdown of ATP6V1E2 in appropriate cell lines

  • Comparison with a second antibody targeting a different epitope of ATP6V1E2

• Loading Controls: Include housekeeping proteins appropriate to the subcellular fraction being analyzed (e.g., GAPDH for cytosolic fractions, Na+/K+ ATPase for membrane fractions)

The control design should account for the antibody's polyclonal nature (all validated options are rabbit polyclonal) by verifying batch-to-batch consistency when continuing long-term research projects .

What are the most common sources of false positives and false negatives when using ATP6V1E2 antibodies, and how can they be mitigated?

False Positives:

• Cross-reactivity with related V-ATPase subunits: ATP6V1E2 shares structural similarities with ATP6V1E1. To mitigate this, use antibodies raised against unique regions (e.g., those targeting amino acids 90-180) and validate with knockout/knockdown controls .

• Non-specific binding in high-background tissues: Certain tissues naturally produce higher background. Optimize by:

  • Increasing blocking duration (3-5% BSA or milk for 1-2 hours)

  • Adding 0.1-0.3% Triton X-100 to blocking buffer

  • Using tissue-specific dilution optimization

  • Extending wash steps (5 × 5 minutes)

False Negatives:

• Epitope masking by fixation: Different fixation methods may mask the target epitope. Compare results between fresh-frozen and formalin-fixed samples when establishing protocols .

• Insufficient protein extraction: ATP6V1E2 may require specialized extraction due to membrane association. Test multiple extraction buffers when working with new tissue types .

• Inappropriate antibody dilution: The recommended ranges (1:500-1:2000) may need adjustment based on specific experimental conditions. Perform dilution series tests when adapting to new experimental systems .

To systematically address both false positive and negative results, implement a multi-antibody approach using antibodies targeting different epitopes of ATP6V1E2 (e.g., comparing antibodies raised against AA 1-226 versus AA 71-120) .

How should researchers interpret discrepancies between calculated (26 kDa) and observed (31-33 kDa) molecular weights of ATP6V1E2?

The discrepancy between the calculated molecular weight (26 kDa based on 226 amino acids) and the observed molecular weight (31-33 kDa) is a significant consideration when interpreting Western blot results . This difference of approximately 5-7 kDa likely results from:

• Post-translational modifications: Phosphorylation sites on ATP6V1E2 can add measurable mass and affect protein migration.

• Glycosylation patterns: Variable glycosylation can significantly impact apparent molecular weight.

• Tissue-specific processing: Different tissues may process ATP6V1E2 differently, resulting in tissue-specific migration patterns.

• Technical factors: SDS-PAGE conditions (gel percentage, buffer systems) can influence migration patterns.

When interpreting results showing bands outside the expected 31-33 kDa range, consider:

• Performing phosphatase treatment of samples to identify phosphorylation contributions

• Using gradient gels to better resolve the protein of interest

• Confirming identity through mass spectrometry when possible

• Comparing migration patterns across multiple tissue/cell types

The observed molecular weight should be consistent with the 31-33 kDa range in validated systems (mouse brain tissue, MCF-7 cells); significant deviations may indicate alternative isoforms, degradation products, or non-specific binding .

What strategies should be employed when ATP6V1E2 antibody performance differs between applications (WB vs. IP vs. IF) in the same experimental system?

Differential performance across applications is a common challenge with ATP6V1E2 antibodies, as epitope accessibility varies by technique. To address this systematically:

• Epitope-dependent considerations:

  • For Western blotting: Denaturing conditions expose linear epitopes, so antibodies recognizing linear sequences (such as those against AA 90-180) typically perform well .

  • For immunoprecipitation: Antibodies must recognize native conformations, requiring those that bind surface-exposed epitopes .

  • For immunofluorescence: Fixation method dramatically affects epitope preservation and accessibility.

• Application-specific optimization:

  • Adjust antibody concentration independently for each application

  • For IF applications showing poor results but strong WB signal, test multiple fixation protocols

  • For IP applications, increase antibody amount (up to 4 μg per reaction) and extend binding time

• Strategic approaches when application differences persist:

  • Use different antibody clones optimized for specific applications

  • Consider alternative detection methods (proximity ligation assay)

  • Implement complementary approaches (mass spectrometry after IP)

  • Validate findings using orthogonal techniques (qPCR correlation with protein levels)

When planning multi-application experiments, anticipate different optimal conditions for each technique and validate each application independently before combining in comprehensive studies .

How can ATP6V1E2 antibodies be effectively utilized in studies of protein-protein interactions within V-ATPase complexes?

For investigating ATP6V1E2's interactions within V-ATPase complexes, researchers should implement a multi-technique approach:

• Co-immunoprecipitation strategies:

  • Use gentler lysis conditions (150-200 mM NaCl, 0.5% NP-40) to preserve protein-protein interactions

  • Perform bidirectional co-IP (using ATP6V1E2 antibody to pull down complexes, then probe for interacting partners; also use antibodies against suspected interacting partners to pull down and probe for ATP6V1E2)

  • Scale antibody amounts to 2-4 μg per reaction when investigating weak or transient interactions

• Proximity-based approaches:

  • Consider BioID or APEX2 proximity labeling using ATP6V1E2 as the bait protein

  • Validate interactions identified through proximity labeling using co-IP with ATP6V1E2 antibodies

• Analytical considerations:

  • Distinguish between direct and indirect interactions through staged IP protocols

  • Map interaction domains using truncated constructs and domain-specific antibodies

  • Compare interaction profiles across different cellular contexts (e.g., different tissues, pH conditions, or ATP availability)

For optimal results, validate interactions under physiologically relevant conditions that preserve V-ATPase complex integrity. Compare findings in mouse brain tissue (where ATP6V1E2 has been well-validated) to establish baseline interaction networks before extending to other experimental systems .

What considerations should researchers address when using ATP6V1E2 antibodies in studies of subcellular localization and trafficking?

Studying ATP6V1E2 subcellular localization presents unique challenges due to its dynamic distribution in various membrane compartments. Researchers should consider:

• Fixation method optimization:

  • Compare paraformaldehyde (preserves structure) with methanol (better epitope accessibility) fixation

  • Optimize permeabilization conditions (0.1-0.3% Triton X-100 or 0.05-0.1% saponin)

  • Consider light fixation followed by detergent extraction for membrane-associated proteins

• Co-localization strategy:

  • Pair ATP6V1E2 antibody with markers for:

    • Lysosomes (LAMP1, LAMP2)

    • Early endosomes (EEA1)

    • Golgi apparatus (GM130)

    • Plasma membrane (Na+/K+ ATPase)

  • Quantify co-localization using appropriate statistical methods (Pearson's coefficient, Manders' overlap)

• Dynamic trafficking studies:

  • Combine antibody labeling with live-cell compatible approaches

  • Consider pulse-chase experiments with surface biotinylation

  • Implement pH-sensitive probes to correlate ATP6V1E2 localization with functional acidification

• Technical refinements:

  • Use super-resolution microscopy (STED, STORM) to resolve small membrane compartments

  • Implement subcellular fractionation followed by Western blotting as a complementary approach

  • Compare results between endogenous labeling (antibody) and tagged constructs

When designing visualization experiments, account for the antibody's specificity profile and validate subcellular localization patterns using multiple fixation and permeabilization protocols to ensure comprehensive characterization .

How can ATP6V1E2 antibodies be integrated into studies investigating V-ATPase dysregulation in disease models?

ATP6V1E2 antibodies provide valuable tools for investigating V-ATPase dysregulation in pathological conditions. For disease model studies:

• Quantitative expression analysis:

  • Implement carefully controlled Western blot protocols with recombinant protein standards for absolute quantification

  • Use quantitative immunohistochemistry with digital image analysis for spatial distribution

  • Compare ATP6V1E2 expression with other V-ATPase subunits to identify subunit-specific dysregulation

• Disease-specific considerations:

  • Neurodegenerative disorders: Focus on ATP6V1E2 expression in brain tissues where positive detection has been validated

  • Cancer models: Compare ATP6V1E2 levels in MCF-7 cells (positive control) with other cancer cell lines

  • Metabolic disorders: Examine expression in relation to metabolic stress conditions

• Functional correlation approaches:

  • Pair ATP6V1E2 antibody detection with functional V-ATPase activity assays

  • Correlate ATP6V1E2 levels with compartmental pH measurements

  • Analyze post-translational modifications using phospho-specific antibodies when available

• Therapeutic targeting assessment:

  • Use ATP6V1E2 antibodies to monitor protein levels following V-ATPase-targeted interventions

  • Implement antibody-based proximity assays to detect conformational changes in response to small molecule modulators

  • Develop cell-based screening approaches using ATP6V1E2 antibodies as readouts

For translation-focused research, validate findings across multiple model systems and correlate with human patient samples when possible to establish clinical relevance of observed ATP6V1E2 alterations .

What emerging techniques can enhance the utility of ATP6V1E2 antibodies beyond traditional applications?

Recent methodological innovations offer expanded research applications for ATP6V1E2 antibodies:

• Multiplexed detection systems:

  • Mass cytometry (CyTOF) using metal-conjugated ATP6V1E2 antibodies for single-cell analysis

  • Multiplexed ion beam imaging (MIBI) for high-parameter tissue analysis

  • Sequential fluorescence detection using antibody stripping and reprobing

• In situ proximity assays:

  • Proximity ligation assay (PLA) to visualize ATP6V1E2 interactions with other V-ATPase subunits

  • Enzyme complementation assays to study dynamic protein associations

  • FRET-based approaches using labeled primary antibodies

• Intrabody applications:

  • Developing intracellularly expressed antibody fragments based on validated ATP6V1E2 antibody sequences

  • Creating nanobody alternatives with improved penetration characteristics

  • Engineering conformation-specific intrabodies to detect structural states

• Tissue clearing compatibility:

  • Validating ATP6V1E2 antibodies in CLARITY, iDISCO, or CUBIC cleared tissue preparations

  • Establishing whole-organ ATP6V1E2 distribution maps using light-sheet microscopy

These approaches extend beyond traditional immunoblotting and immunoprecipitation to provide spatial, temporal, and interaction data previously inaccessible with conventional antibody applications. When implementing these advanced methods, researchers should include appropriate controls adapted to each specific technology platform .

How should researchers approach antibody validation when studying ATP6V1E2 in non-standard experimental models or rare tissues?

When extending ATP6V1E2 research to non-validated models or rare tissues, implement a systematic validation approach:

• Hierarchical validation strategy:

  • Begin with in silico analysis: Confirm target sequence homology across species

  • Perform epitope mapping: Identify if the immunizing sequence (e.g., AA 90-180 or 1-226) is conserved in the model organism

  • Establish baseline expression: Use quantitative PCR to confirm ATP6V1E2 transcript presence before protein analysis

• Cross-validation methodology:

  • Use multiple antibodies targeting different ATP6V1E2 epitopes

  • Compare commercial antibodies from different vendors (Proteintech, Novus, Atlas Antibodies)

  • Implement genetic approaches (siRNA, CRISPR) to confirm specificity when possible

• Tissue-specific optimization:

  • Develop tissue-specific protein extraction protocols

  • Optimize fixation parameters for immunohistochemistry applications

  • Establish appropriate positive controls specific to the tissue type

• Documentation standards:

  • Record batch-specific validation data

  • Document all optimization steps systematically

  • Share validation protocols through repositories or supplementary materials

For extremely rare tissues or specialized research models, consider preparing customized validation standards by expressing recombinant ATP6V1E2 matching the species of interest, then using this as a defined positive control for antibody performance assessment .

What considerations should researchers address when designing longitudinal studies that require consistent ATP6V1E2 antibody performance across extended timeframes?

Longitudinal studies face unique challenges regarding antibody consistency. To ensure reliable ATP6V1E2 detection across extended research timelines:

• Antibody management strategy:

  • Purchase sufficient antibody from a single lot for the entire study duration

  • Create standardized aliquots with consistent antibody concentration

  • Store reference aliquots unopened until needed for validation

  • Document lot numbers and maintain certificate of analysis information

• Performance monitoring protocol:

  • Implement regular validation checkpoints using consistent positive controls

  • Develop quantitative metrics for antibody performance (signal-to-noise ratio)

  • Establish acceptance criteria before study initiation

  • Create a decision tree for troubleshooting if performance declines

• Technical standardization:

  • Standardize all experimental conditions (buffers, incubation times, temperatures)

  • Prepare master mixes of recurring reagents when possible

  • Implement automated protocols where feasible to reduce variability

  • Include internal reference standards in each experiment

• Contingency planning:

  • Identify alternative ATP6V1E2 antibodies validated for the same applications

  • Maintain protocols for comparing performance between primary and backup antibodies

  • Consider developing custom antibodies for critical research programs