ATP6AP2 Antibody

What is ATP6AP2 and why is it important in research?

ATP6AP2 (ATPase H⁺ transporting accessory protein 2) is an essential component of the vacuolar H⁺-ATPase that functions in numerous cellular processes. It was originally identified as the (pro)renin receptor but has since been recognized for its broader role in multiple biological systems. ATP6AP2 is critical for basic cellular mechanisms and necessary for multiple organ function . Research interest in ATP6AP2 stems from its involvement in development, hematopoiesis, metabolism, cell proliferation, and its potential as a therapeutic target for various diseases .

What are the known subcellular localizations of ATP6AP2?

ATP6AP2 has been detected in multiple subcellular compartments. Overexpression studies using the wing driver patched (ptc)-GAL4 have shown strong colocalization with the endoplasmic reticulum (ER) marker protein disulfide-isomerase (PDI) . Native ATP6AP2 has also been detected in intracellular pools, though these signals may be weaker than with overexpression methods . In the kidney, ATP6AP2 colocalizes with H⁺-ATPase subunit a4 in proximal tubule and intercalated cells .

What forms of ATP6AP2 can be detected with antibodies?

ATP6AP2 can be detected in multiple forms due to post-translational processing. Antibodies can detect the full-length (FL) protein as well as cleaved fragments produced in the Golgi apparatus . When selecting an antibody, researchers should consider which form(s) they wish to detect and choose antibodies with appropriate epitope recognition.

What are the validated protocols for ATP6AP2 immunohistochemistry?

For immunohistochemical detection of ATP6AP2, the following validated protocol has been used:

• Perform microwave antigen retrieval in 10 mM sodium citrate buffer (pH 6.0)

• Dilute rabbit anti-ATP6AP2 antisera (such as Sigma HPA003156) at 1/500 in PBS containing 0.05% Tween-20

• Block sections with 10% normal horse serum and 0.3% Triton X-100

• Detect primary antibody by incubating sections for 1 hour at room temperature with Alexa Fluor 555-conjugated secondary antibody (1:500, Invitrogen Molecular Probes)

• Mount sections with Mowiol 4-88 mounting medium containing DAPI (5 μg/ml)

This protocol has been validated in previous studies and provides specific detection of ATP6AP2 in tissue sections.

How can ATP6AP2 protein expression levels be quantified accurately?

ATP6AP2 protein expression can be quantified using standard immunoblotting techniques. Studies have shown that both wild-type and mutant ATP6AP2 (such as L98S and R71H) can be detected and quantified using appropriate antibodies . For accurate quantification:

• Use well-validated antibodies against ATP6AP2

• Include appropriate loading controls

• Perform densitometric analysis of multiple blots

• Compare results with mRNA quantification (RT-qPCR) to account for post-transcriptional regulation

Research has demonstrated that mutations can affect steady-state protein levels, with some mutations like L98S showing reduced protein levels while maintaining normal mRNA expression, suggesting post-translational regulation .

What controls should be included when using ATP6AP2 antibodies?

When designing experiments using ATP6AP2 antibodies, include the following controls:

• Positive controls: Tissues or cells known to express ATP6AP2 (e.g., kidney samples)

• Negative controls: Samples with ATP6AP2 knockdown or knockout (when available)

• Technical controls: Primary antibody omission to assess background staining

• Validation controls: Comparison of results with multiple antibodies targeting different epitopes

• Expression controls: Comparison of protein detection with mRNA quantification

For knockdown models, transgenic rats expressing shRNA against ATP6AP2 have shown approximately 90% reduction in ATP6AP2 mRNA and protein and can serve as useful controls for antibody specificity .

How can ATP6AP2 antibodies be used to study its role in the kidney?

ATP6AP2 antibodies are valuable tools for investigating its role in kidney function. Studies have demonstrated that ATP6AP2 is involved in receptor-mediated endocytosis in proximal tubules . Research approaches include:

• Immunohistochemistry: Co-staining of ATP6AP2 with H⁺-ATPase subunit a4 to examine localization in proximal tubule and intercalated cells

• Loss-of-function studies: Analysis of kidney phenotypes in ATP6AP2 knockdown models

• Functional assays: Assessment of receptor-mediated endocytosis in models with altered ATP6AP2 expression

Experiments in transgenic rats with ATP6AP2 knockdown have revealed mild impairment of renal function, with elevated BUN and blood phosphate levels, decreased creatinine clearance, and altered urinary electrolyte excretion as shown in the following table :

What approaches are available to study the relationship between ATP6AP2 and V-ATPase assembly?

ATP6AP2 functions as a V-ATPase assembly factor . To investigate this relationship:

• Co-immunoprecipitation: Using ATP6AP2 antibodies to pull down V-ATPase components

• Proximity labeling: BioID or APEX-based approaches to identify spatial relationships

• Subcellular fractionation: Combined with immunoblotting to examine co-localization in cellular compartments

• Functional assays: Measuring V-ATPase activity in ATP6AP2-deficient cells

• Structural studies: Examining how ATP6AP2 interacts with V-ATPase components

These approaches can provide insights into the molecular mechanisms by which ATP6AP2 facilitates V-ATPase assembly and function.

How can ATP6AP2 antibodies help in studying hematopoietic disorders?

ATP6AP2 plays a critical role in hematopoiesis, with its ablation causing severe leukopenia and bone marrow hypoplasia . Antibodies can be used to:

• Characterize ATP6AP2 expression: Examine ATP6AP2 levels in different hematopoietic cell populations

• Study ATP6AP2 in HSC self-renewal: Investigate its relationship with WNT signaling

• Analyze pathological samples: Compare ATP6AP2 expression in normal versus diseased tissues

• Monitor therapeutic responses: Assess ATP6AP2 levels following treatment

Research has shown that ATP6AP2 deficiency results in an 80-90% decrease in peripheral blood leukocytes affecting all lineages, and defects in bone marrow progenitors, indicating its essential role in maintaining hematopoietic stem cells .

What factors can influence ATP6AP2 antibody specificity and sensitivity?

Several factors can affect the performance of ATP6AP2 antibodies:

• Epitope accessibility: ATP6AP2 undergoes cleavage in the Golgi apparatus, which may affect epitope availability

• Cross-reactivity: Some antibodies may recognize related proteins

• Fixation conditions: Different fixatives can affect antigen recognition

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications may alter antibody binding

• Expression levels: Endogenous ATP6AP2 may be expressed at low levels, requiring sensitive detection methods

Optimization of sample preparation and detection protocols is essential for obtaining specific and sensitive results with ATP6AP2 antibodies.

How can contradictory results with ATP6AP2 antibodies be resolved?

When facing contradictory results with ATP6AP2 antibodies, consider the following approaches:

• Use multiple antibodies: Employ antibodies targeting different epitopes of ATP6AP2

• Validate with alternative methods: Confirm findings using mRNA quantification, overexpression systems, or knockdown models

• Consider tissue-specific effects: ATP6AP2 may have different expression patterns and functions in various tissues

• Examine technical variables: Assess the impact of sample preparation, fixation, and detection methods

• Account for genetic background: ATP6AP2 function may vary depending on genetic context

Research has demonstrated that different mutations in ATP6AP2 can have varying effects on protein stability and mRNA levels, which could explain some contradictory findings .

How can ATP6AP2 antibodies be used to study its role in WNT signaling pathways?

ATP6AP2 has been implicated in WNT signaling, particularly in hematopoietic stem cell (HSC) self-renewal . Advanced research approaches include:

• Chromatin immunoprecipitation (ChIP): Using ATP6AP2 antibodies to identify potential transcriptional targets

• Proximity-dependent biotinylation: Identifying ATP6AP2 interaction partners in WNT signaling

• Single-cell approaches: Examining ATP6AP2 expression and function in individual cells

• Conditional knockout models: Studying tissue-specific effects of ATP6AP2 deletion on WNT pathway components

• Phospho-specific antibodies: Investigating post-translational modifications of ATP6AP2 in response to WNT stimulation

These approaches can provide mechanistic insights into how ATP6AP2 contributes to WNT signaling and HSC maintenance.

What is the relationship between ATP6AP2 mutations and glycosylation disorders?

Mutations in X-linked ATP6AP2 have been reported to cause glycosylation disorders with liver disease, immunodeficiency, and psychomotor impairment . To investigate this relationship:

• Structural studies: Analyzing how mutations affect ATP6AP2 protein structure and stability

• Functional assays: Measuring glycosylation efficiency in cells with ATP6AP2 mutations

• Protein-protein interaction studies: Examining how mutations affect ATP6AP2's interactions with glycosylation machinery

• Animal models: Generating knock-in models of human ATP6AP2 mutations to study pathophysiology

• Clinical correlations: Analyzing the relationship between specific mutations and clinical manifestations

Research has shown that different mutations can have distinct effects on ATP6AP2 protein levels and mRNA expression. For example, the L98S mutation significantly reduces steady-state levels of full-length ATP6AP2 without affecting mRNA levels, while the R71H mutation increases mRNA levels but decreases protein expression upon overexpression .

How might post-translational modifications of ATP6AP2 affect antibody recognition?

ATP6AP2 undergoes various post-translational modifications that could impact antibody binding:

• Proteolytic processing: ATP6AP2 is cleaved into fragments in the Golgi apparatus, potentially affecting epitope availability

• Glycosylation: Modifications of N-linked glycosylation sites could alter antibody recognition

• Phosphorylation: Signaling-dependent phosphorylation might induce conformational changes

• Ubiquitination: Protein degradation signals could affect stability and detection

• Conformation-dependent epitopes: Some antibodies may recognize specific protein conformations

For comprehensive analysis, researchers should employ multiple antibodies targeting different regions and consider how sample preparation might affect post-translational modifications.