ATP6V1B2 Antibody

What is ATP6V1B2 and what cellular functions does it regulate?

ATP6V1B2 is a non-catalytic subunit of the V1 complex of vacuolar H⁺-ATPase (V-ATPase), a multisubunit enzyme composed of a peripheral V1 complex that hydrolyzes ATP and a membrane-integral V0 complex that translocates protons. This protein plays a major role in:

• Acidification and pH maintenance of intracellular compartments

• Lysosomal function and autophagy regulation

• Proton transport across cellular membranes

V-ATPase functions as a heteromultimeric enzyme with the V1 complex (including ATP6V1B2) attached to the V0 membrane proton pore complex. ATP6V1B2 is one of two isoforms of ATP6V1 and is expressed in most cell types, displaying a broader expression pattern than ATP6V1B1, which is primarily kidney-specific .

How do ATP6V1B2 antibodies perform across different experimental applications?

ATP6V1B2 antibodies show varying performance characteristics across experimental platforms:

These applications have been validated through published research, with certain antibodies being cited in multiple studies focusing on ATP6V1B2's role in lysosomal function and disease models .

What are the critical steps for successful Western blotting with ATP6V1B2 antibodies?

For optimal Western blot results with ATP6V1B2 antibodies:

• Sample preparation: Efficiently lyse cells using buffers containing protease inhibitors to prevent degradation of the target protein

• Loading control selection: GAPDH is commonly used for normalization when studying ATP6V1B2

• Gel percentage: Use 10-12% SDS-PAGE gels for optimal resolution of the 56-58 kDa ATP6V1B2 protein

• Blocking conditions: 5% non-fat milk or BSA in TBST typically provides adequate blocking

• Primary antibody incubation: Dilute according to manufacturer recommendations (typically 1:1000-1:5000) and incubate overnight at 4°C

• Detection method: Both chemiluminescence and fluorescence-based detection systems work well

• Expected results: The observed molecular weight should be approximately 56-58 kDa

When troubleshooting, note that 293T cells, HeLa cells, mouse brain tissue, and other tissue lysates have been successfully used to detect ATP6V1B2 expression .

How should I design co-localization studies to investigate ATP6V1B2's role in lysosomal function?

To effectively study ATP6V1B2's co-localization with lysosomes:

• Cell preparation: Seed primary fibroblasts or relevant cell lines at 20 × 10³ cells on cover glasses in 24-well plates

• Fixation protocol: Fix cells with 3% PFA for 30 minutes at 4°C

• Permeabilization: Treat with 0.5% Triton X-100 for 10 minutes at room temperature

• Primary antibodies: Use mouse monoclonal anti-LAMP1 antibody as a lysosomal marker alongside rabbit polyclonal ATP6V1B2 antibody

• Secondary antibody selection: Choose fluorophores with minimal spectral overlap

• Counterstaining: DAPI for nuclear visualization

• Imaging: Confocal microscopy is preferable for detailed co-localization analysis

• Analysis: Quantify co-localization using Pearson's or Mander's coefficients

This approach has been used successfully to demonstrate ATP6V1B2's association with lysosomes and document morphological changes in lysosomes in patient-derived fibroblasts carrying ATP6V1B2 mutations .

How do mutations in ATP6V1B2 contribute to neurodevelopmental disorders?

ATP6V1B2 mutations have been linked to several syndromic disorders through distinct molecular mechanisms:

• Dominant deafness-onychodystrophy (DDOD) syndrome: Characterized by congenital sensorineural hearing loss and nail abnormalities

• DOORS syndrome: Features deafness, onychodystrophy, osteodystrophy, intellectual disability, and seizures

• Zimmermann-Laband syndrome (ZLS): Manifests with gingival enlargement, hypoplasia/aplasia of nails and terminal phalanges, and intellectual disability

Recent research demonstrates that these disorders form a phenotypic continuum rather than distinct entities. Dominantly acting variants in ATP6V1B2 (p.Ala332Val, p.Gln376Lys, p.Tyr328His, p.Arg485Pro) result in a gain-of-function mechanism that upregulates V-ATPase function, leading to:

• Increased lysosomal acidification

• Disrupted lysosomal morphology and function

• Defective autophagic flux

• Accumulation of lysosomal substrates

• Impaired cilium biogenesis

These insights have led to the reclassification of these conditions as lysosomal disorders, expanding our understanding of how ATP6V1B2 dysfunction impacts cellular homeostasis.

What experimental models are available for studying ATP6V1B2-related hearing loss?

Several experimental models have been developed to study ATP6V1B2-related hearing loss:

• Hair cell-specific knockout mouse (Atp6v1b2 fl/fl; Atoh1): Recapitulates human phenotypes including hair cell loss and abnormal lysosomal morphology and function

• Atp6v1b2 Arg506/Arg506 mice**: Develops progressive hearing loss starting at 28 weeks with increased ABR thresholds

• Cell culture models: Patient-derived fibroblasts and transfected cell lines expressing mutant ATP6V1B2 constructs

Key findings from these models include:

• ATP6V1B2 is essential for maintaining lysosomal function in hair cells

• Hair cell degeneration can be prevented through gene therapy approaches

• ATP6V1B2 plays a critical role in spiral ganglion neuron maintenance

• Hidden hearing loss (HHL) precedes overt hearing loss in some models

The Arg506*/Arg506* Atp6v1b2 mice show accumulation of autophagosomes in spiral ganglion neurons but not in the organ of Corti, suggesting differential effects of ATP6V1B2 dysfunction across auditory tissues .

How can I effectively use ATP6V1B2 antibodies in immunoprecipitation studies?

For successful immunoprecipitation of ATP6V1B2:

• Lysate preparation: Use 1.0-3.0 mg of total protein lysate from tissues expressing ATP6V1B2 (e.g., mouse brain tissue)

• Antibody amount: 0.5-4.0 μg of purified ATP6V1B2 antibody

• Pre-clearing step: Pre-clear lysate with protein A/G beads to reduce non-specific binding

• Immunoprecipitation: Incubate pre-cleared lysate with antibody overnight at 4°C

• Bead selection: Protein G works well for most ATP6V1B2 antibodies

• Washing conditions: Use stringent washing to minimize non-specific interactions

• Elution method: Gentle elution under non-denaturing conditions can preserve protein-protein interactions

• Controls: Include IgG control and input samples for validation

IP can be followed by mass spectrometry to identify ATP6V1B2 interaction partners or Western blotting to confirm successful pulldown .

What considerations are important when using gene therapy approaches for ATP6V1B2-related disorders?

Recent advancements in gene therapy for ATP6V1B2-related disorders reveal several critical considerations:

• Vector design: Adeno-associated virus (AAV) vectors incorporating inner ear-specific promoters show promise

• Administration route: Direct injection into the scala media at postnatal days 0-2 has demonstrated efficacy

• Expression optimization: The AAV-ie-Eh3 vector system enhances therapeutic precision while minimizing toxicity

• Dose determination: Single administration can provide long-term (24+ weeks) hearing rescue

• Outcome measures:

  • Prevention of hair cell degeneration

  • Restoration of lysosome morphology

  • Rescue of auditory and vestibular function

• Timing considerations: Early intervention before irreversible cellular damage occurs is critical

This approach establishes a novel therapeutic paradigm with significant clinical potential for ATP6V1B2-associated hearing loss and vestibular dysfunction .

How can I confirm the specificity of my ATP6V1B2 antibody?

Confirming ATP6V1B2 antibody specificity requires multiple validation approaches:

• Western blot analysis: Verify the detection of a single band at the expected molecular weight (56-58 kDa)

• Multiple tissue/cell line testing: Test across tissues known to express ATP6V1B2 (brain, kidney, etc.)

• Knockout/knockdown controls: Use ATP6V1B2 knockout or siRNA-treated samples as negative controls

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to confirm signal specificity

• Cross-reactivity assessment: Test against ATP6V1B1 to ensure no cross-reactivity with this related isoform

• Orthogonal validation: Compare results using antibodies targeting different epitopes of ATP6V1B2

• Enhanced validation: Some antibodies undergo orthogonal RNAseq validation, providing additional confidence

Antibody validation data should show reactivity with the expected species (human, mouse, rat) and appropriate subcellular localization consistent with ATP6V1B2's known distribution.

What factors affect experimental variability when working with ATP6V1B2 antibodies?

Several factors can introduce variability in ATP6V1B2 antibody experiments:

• Antibody storage conditions: Store at -20°C or -80°C according to manufacturer recommendations; avoid repeated freeze-thaw cycles

• Buffer composition: Use recommended buffers (PBS with 0.02% sodium azide and 50% glycerol pH 7.3 for many antibodies)

• Sample preparation inconsistencies: Standardize lysis protocols and protein quantification methods

• Application-specific factors:

  • WB: Transfer efficiency, blocking conditions, antibody concentration

  • IHC: Fixation method, antigen retrieval (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • IF: Cell permeabilization, mounting media selection

• Antibody class differences: Monoclonal vs. polyclonal antibodies may show different specificities and applications

• Cell/tissue-specific expression levels: ATP6V1B2 expression varies across tissues, affecting detection sensitivity

• Post-translational modifications: May affect epitope recognition

To minimize variability, careful titration of antibody concentration for each experimental system and thorough documentation of protocols are essential.