ATP6AP1L Antibody

What is ATP6AP1L and why is it important in research?

ATP6AP1L (ATPase H+ transporting accessory protein 1 like) is a human protein also known as V-type proton ATPase subunit S1-like protein. It has a molecular weight of approximately 25.3 kilodaltons . This protein is structurally related to ATP6AP1, which functions as an accessory subunit of the proton-transporting vacuolar (V)-ATPase protein pump. While ATP6AP1 has been well-characterized in the regulation of luminal acidification of secretory vesicles, the specific functions of ATP6AP1L are still being elucidated, making it an important target for research into cellular pH regulation and membrane trafficking mechanisms.

What applications are ATP6AP1L antibodies most commonly used for?

ATP6AP1L antibodies are primarily used for Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunofluorescence (IF), and Immunohistochemistry (IHC) . The application distribution varies by supplier and antibody clone. For instance, some antibodies are specifically optimized for Western Blotting, while others may be better suited for immunohistochemical applications. Researchers should carefully evaluate manufacturer specifications to select the appropriate antibody for their specific application needs.

What species reactivity is available for ATP6AP1L antibodies?

Most commercially available ATP6AP1L antibodies demonstrate reactivity with human ATP6AP1L, with some showing cross-reactivity with orthologs from various species including dog (93% predicted reactivity), horse (92%), and pig (79%) . When working with non-human models, it's essential to verify the cross-reactivity through alignment of the immunogen sequence with the target species' protein sequence.

How does ATP6AP1L differ functionally from ATP6AP1, and what implications does this have for antibody selection?

While both proteins share structural similarities, ATP6AP1 has been identified as the functional ortholog of yeast V-ATPase assembly factor Voa1 and plays a crucial role in V-ATPase assembly and regulation . ATP6AP1 deficiency has been linked to immunodeficiency with hepatopathy and neurocognitive abnormalities .

In contrast, the specific functions of ATP6AP1L remain less characterized. When selecting antibodies, researchers should ensure specificity for either ATP6AP1L or ATP6AP1 by examining the epitope recognition site. Antibodies targeting the C-terminal region of ATP6AP1L, such as those recognizing amino acids 190-209 , can help ensure specificity. Cross-reactivity testing is strongly recommended when studying these proteins in parallel.

What are the challenges in detecting different tissue-specific isoforms of ATP6AP1L?

Based on the knowledge of ATP6AP1, which exists in different isoforms across tissues (62-kDa intact protein in liver, 40-kDa processed form in brain, and 50-kDa isoform in B-cells) , it's reasonable to hypothesize that ATP6AP1L might also exhibit tissue-specific processing.

When designing experiments to detect potential ATP6AP1L isoforms:

• Use antibodies targeting different epitopes (N-terminal, C-terminal, and middle regions)

• Include positive controls from different tissue types

• Optimize protein extraction methods to preserve potential post-translational modifications

• Consider using both reducing and non-reducing conditions for Western blotting

• Validate findings with mass spectrometry to confirm isoform identity

What are the optimal conditions for using ATP6AP1L antibodies in Western blotting?

For Western blotting applications with ATP6AP1L antibodies:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

• Gel electrophoresis:

  • 10-12% SDS-PAGE gels are typically suitable for the 25.3 kDa ATP6AP1L protein

  • Transfer to PVDF membranes (preferred over nitrocellulose for this protein)

• Antibody incubation:

  • Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Primary antibody: Start with 1:1000 dilution and optimize as needed

  • Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5000 dilution is recommended for most ATP6AP1L polyclonal rabbit antibodies

• Detection:

  • Enhanced chemiluminescence (ECL) is suitable for most applications

  • Expected band size: ~25.3 kDa, but verify potential tissue-specific isoforms

What controls should be included when using ATP6AP1L antibodies in immunohistochemistry?

For rigorous IHC experiments with ATP6AP1L antibodies:

• Positive controls:

  • Human tissues with known ATP6AP1L expression

  • Cell lines with confirmed ATP6AP1L expression

• Negative controls:

  • Primary antibody omission

  • Isotype control antibody

  • Pre-absorption of the antibody with the immunizing peptide

• Specificity controls:

  • Tissues from knockout models (if available)

  • Samples with ATP6AP1L knockdown by siRNA

  • Comparative staining with multiple antibodies targeting different epitopes

• Protocol optimization:

  • Test multiple antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0)

  • Titrate antibody concentration (starting from manufacturer's recommendation)

  • Optimize incubation time and temperature

How can researchers distinguish between ATP6AP1L and ATP6AP1 in experimental results?

Distinguishing between these related proteins requires careful experimental design:

• Antibody selection:

  • Use antibodies targeting unique epitopes specific to either protein

  • Verify specificity through sequence alignment of the immunogen

• Molecular weight differentiation:

  • ATP6AP1L: Expected at approximately 25.3 kDa

  • ATP6AP1: Exists in multiple forms (40-62 kDa depending on tissue)

• Expression pattern analysis:

  • ATP6AP1 shows tissue-specific isoforms with distinct sizes

  • Compare expression patterns across tissues to identify distinctive profiles

• Functional validation:

  • ATP6AP1 has established roles in V-ATPase assembly and lysosomal acidification

  • Knockdown/knockout experiments can help confirm target identity based on functional phenotypes

• Double immunostaining:

  • Co-staining with verified antibodies against both proteins can reveal distinct or overlapping localization patterns

What are common pitfalls when interpreting ATP6AP1L antibody results in the context of disease models?

When studying ATP6AP1L in disease contexts, researchers should be aware of:

• Cross-reactivity concerns:

  • ATP6AP1 has established connections to colorectal cancer , immunodeficiency , and breast cancer

  • Confirm that observed disease associations are specific to ATP6AP1L and not due to cross-reactivity

• Expression level interpretation:

  • Changes in ATP6AP1L expression might reflect altered V-ATPase activity rather than direct disease causation

  • Validate functional significance through mechanistic studies

• Isoform-specific effects:

  • Different tissues may express different isoforms with distinct functions

  • Disease-related changes might affect specific isoforms only

• Context-dependent functions:

  • The role of ATP6AP1L might vary based on cell type, tissue, or disease state

  • Control experiments should match the specific context being studied

How can researchers design experiments to investigate potential functional overlap between ATP6AP1L and ATP6AP1?

To explore functional relationships between these proteins:

What methodological approaches are recommended for investigating ATP6AP1L in immune cell function, based on what is known about ATP6AP1?

Given that ATP6AP1 deficiency causes immunodeficiency , exploring ATP6AP1L's role in immune function requires:

• Cell type-specific expression analysis:

  • Characterize ATP6AP1L expression across immune cell subsets using flow cytometry

  • Compare with known ATP6AP1 expression patterns

• Functional immune assays:

  • B cell activation and antibody production

  • T cell activation and cytokine secretion

  • Antigen presentation by dendritic cells

  • Phagocytosis by macrophages

• V-ATPase-dependent processes:

  • Lysosomal acidification in immune cells

  • MHC class II antigen processing

  • Toll-like receptor signaling

• In vivo models:

  • Conditional knockout in specific immune cell populations

  • Challenge with pathogens or immunization protocols

  • Monitor humoral and cellular immune responses

How might ATP6AP1L be involved in cancer biology, and what experimental approaches would best investigate this?

Based on ATP6AP1's role in colorectal cancer and breast cancer , potential roles for ATP6AP1L in cancer warrant investigation:

• Expression analysis:

  • Comprehensive screening across cancer types using tissue microarrays

  • Correlation with clinical outcomes and tumor characteristics

• Functional cancer hallmarks assessment:

  • Proliferation after ATP6AP1L knockdown/overexpression

  • Migration and invasion assays

  • Anchorage-independent growth

  • Drug resistance phenotypes

• Mechanism exploration:

  • Impact on autophagy (similar to ATP6AP1's role in breast cancer )

  • Effects on lysosomal function in cancer cells

  • Alterations in cellular pH regulation

  • Changes in protein trafficking and degradation

• Therapeutic implications:

  • Potential as a biomarker (similar to ATP6AP1 in colorectal cancer )

  • Vulnerability to V-ATPase inhibitors

  • Combination approaches with standard therapies

What are the recommended approaches for developing novel and more specific ATP6AP1L antibodies for research use?

To develop improved ATP6AP1L antibodies:

• Epitope selection:

  • Target unique regions with minimal homology to ATP6AP1

  • Consider both linear and conformational epitopes

  • Utilize structural biology data if available

• Production strategies:

  • Recombinant antibody technology for reproducibility

  • Phage display for high-affinity selection

  • Monoclonal approaches for specificity

• Validation requirements:

  • Testing in multiple sample types (cell lines, tissues, recombinant proteins)

  • Knockout/knockdown controls

  • Cross-reactivity assessment against ATP6AP1

  • Application-specific validation (WB, IF, IHC, IP)

• Emerging technologies:

  • Nanobodies for improved tissue penetration

  • Bispecific antibodies for co-localization studies

  • Proximity labeling applications