ATP6AP1 Antibody

What is ATP6AP1 and what is its primary cellular function?

ATP6AP1, also known as Ac45, is a type I transmembrane accessory protein of the V-ATPase complex. The protein is predominantly expressed in endocrine and neuronal cells and plays a critical role in targeting the V-ATPase enzyme to specialized vacuolar systems . Through its cytoplasmic tail, ATP6AP1 interacts with the V0 domain subunits of V-ATPase . This interaction is essential for the acidification of eukaryotic intracellular organelles, which facilitates various cellular processes including protein degradation, nutrient storage, and pH regulation . In plants, ATP6AP1 creates an electrochemical gradient within biological membranes that serves as an energy source for metabolite uptake and environmental responses .

What applications are ATP6AP1 antibodies suitable for?

ATP6AP1 antibodies have been validated for multiple research applications:

• Western Blotting (WB): Used to detect ATP6AP1 protein expression levels in tissue/cell lysates

• Immunofluorescence (IF): Allows visualization of ATP6AP1 subcellular localization

• Immunohistochemistry (IHC): Enables detection of ATP6AP1 in tissue sections

• Immunocytochemistry (ICC): Used to identify ATP6AP1 in cultured cells

• Enzyme-Linked Immunosorbent Assay (ELISA): Quantitative measurement of ATP6AP1

• Immunoprecipitation (IP): Isolation of ATP6AP1 and associated protein complexes

• Immunolocalization (IL): Precise localization of ATP6AP1 within cellular compartments

Selection of the appropriate application depends on your specific research questions and experimental design. For complex tissue analyses, a combination of methods may provide more comprehensive results.

What species reactivity do commercially available ATP6AP1 antibodies exhibit?

Commercial ATP6AP1 antibodies demonstrate reactivity across multiple species. Based on the search results, the following reactivity profiles are available:

• Human-specific antibodies: Several monoclonal and polyclonal options target human ATP6AP1

• Mouse and rat cross-reactive antibodies: Important for comparative mammalian studies

• Plant-specific antibodies: Some antibodies show reactivity with Arabidopsis thaliana, Chlamydomonas, and Soybean (Glycine max)

When selecting an antibody, it's important to verify the targeted epitope region, as some antibodies recognize specific domains (e.g., AA 51-150, AA 90-390, or AA 421-470) which may affect cross-reactivity and specificity . For evolutionarily conserved regions of ATP6AP1, cross-reactivity is more likely, while antibodies targeting variable regions may offer species specificity.

How should researchers optimize Western blotting protocols for ATP6AP1 detection?

For optimal Western blotting results with ATP6AP1 antibodies, consider the following methodological recommendations:

First, sample preparation is critical. For ATP6AP1 detection in Arabidopsis thaliana, the expected molecular weight is approximately 95 kDa . In human samples, prepare lysates with a protease inhibitor cocktail to prevent degradation of the target protein. Use RIPA or NP-40 buffer for membrane protein extraction, as ATP6AP1 is a transmembrane protein.

For electrophoresis and transfer:

• Use 8-10% SDS-PAGE gels for optimal separation

• Include positive controls from tissues known to express ATP6AP1 (e.g., brain tissues or cell lines with confirmed expression)

• Transfer to PVDF membranes, which often provide better results for transmembrane proteins

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

For antibody incubation:

• Dilute primary antibodies appropriately (typically 1:500 to 1:1000) based on manufacturer recommendations

• Incubate overnight at 4°C for optimal binding

• Use the appropriate secondary antibody conjugated to HRP

• Consider using m-IgG Fc BP-HRP or m-IgGκ BP-HRP bundles for enhanced sensitivity

Validate results by including appropriate controls and consider using ATP6AP1 antibodies from different clones that recognize distinct epitopes to confirm specificity.

What are the key considerations when using ATP6AP1 antibodies for studying its role in immune cell infiltration?

When investigating ATP6AP1's relationship with immune cell infiltration, particularly in colorectal cancer research, several methodological considerations are important:

First, tissue preparation is crucial. Use fresh-frozen or appropriately fixed tissues to preserve both ATP6AP1 expression and immune cell markers. For dual immunofluorescence staining, optimize antigen retrieval methods that work effectively for both ATP6AP1 and immune cell markers.

Second, select appropriate immune cell markers to study correlations:

• For dendritic cells (iDC): CD11c, CD83

• For regulatory T cells (TReg): FOXP3, CD25

• For cytotoxic T cells: CD8

• For B cells: CD20

• For macrophages: CD68

Research has demonstrated significant correlations between ATP6AP1 expression and infiltration of specific immune cell populations. In rectal cancer (READ), ATP6AP1 expression positively correlates with infiltration of dendritic cells (R = 0.27, p < 0.001), eosinophils (R = 0.224, p = 0.004), and regulatory T cells (R = 0.328, p < 0.001) . These correlations provide potential research directions for immunomodulatory mechanisms.

Additionally, when analyzing ATP6AP1's relationship with immune infiltration, consider using computational approaches such as single-sample Gene Set Enrichment Analysis (ssGSEA) alongside experimental validation. The TIMER2 and TISIDB databases can be valuable resources for initial in silico assessments of these relationships .

How do monoclonal and polyclonal ATP6AP1 antibodies compare in research applications?

Monoclonal and polyclonal ATP6AP1 antibodies each offer distinct advantages for different research applications:

Monoclonal ATP6AP1 Antibodies:

• Provide high specificity for a single epitope

• Examples include clone 85.1 (mouse monoclonal IgG1 κ) targeting human ATP6AP1 and clone E-10

• Offer consistent lot-to-lot reproducibility

• Ideal for applications requiring precise epitope targeting

• Better suited for quantitative analyses and therapeutic development

• May be less effective if the target epitope is masked or modified

Polyclonal ATP6AP1 Antibodies:

• Recognize multiple epitopes on ATP6AP1

• Examples include rabbit polyclonal antibodies targeting internal regions (AA 90-390) or specific domains

• Provide stronger signal due to binding of multiple antibodies per target molecule

• Better for detecting denatured proteins or modified forms

• Useful for initial characterization studies

• Show higher batch-to-batch variation

How can ATP6AP1 antibodies be used to investigate its potential as a biomarker for colorectal cancer?

Recent research has identified ATP6AP1 as a promising biomarker for colorectal cancer (CRC) prognosis. To investigate this potential, researchers should consider the following methodological approaches using ATP6AP1 antibodies:

First, perform comparative immunohistochemistry using tissue microarrays containing both CRC and matched normal tissues. Recent studies have demonstrated that ATP6AP1 expression is significantly elevated in CRC and associated with poor clinicopathological characteristics and prognosis . Using standardized scoring systems (H-score or IHC score), quantify ATP6AP1 expression and correlate with clinical parameters.

Second, conduct survival analyses by stratifying patients based on ATP6AP1 expression levels. According to The Cancer Genome Atlas (TCGA) data analysis, high ATP6AP1 expression correlates with poorer prognosis in CRC patients . This approach can help validate ATP6AP1 as a prognostic marker in independent cohorts.

Third, investigate ATP6AP1's relationship with immune checkpoints. Recent findings indicate significant correlations between ATP6AP1 expression and immune checkpoint molecules including TNFRSF8, CD40, PDCD1, TNFRSF18, TNFRSF25, TNFRSF4, TNFRSF14, VSIR, CD276, VTCN1, and BTLA in CRC (all p < 0.05) . These correlations suggest ATP6AP1 may influence immunosuppression in the CRC microenvironment, making it relevant to immunotherapy response prediction.

Finally, examine ATP6AP1's association with cancer-associated fibroblasts (CAFs). Significant positive correlations have been found between ATP6AP1 expression and CAF markers in both colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) . This suggests a potential role in tumor-stromal interactions that could impact therapeutic approaches.

What methodologies are effective for studying ATP6AP1's role in immunodeficiency disorders?

ATP6AP1 deficiency has been linked to an immunodeficiency with hepatopathy, highlighting its importance in immune system function . When investigating ATP6AP1's role in immunodeficiency disorders, consider these methodological approaches:

First, perform comprehensive protein expression analysis. Use ATP6AP1 antibodies for Western blotting and immunohistochemistry to compare expression levels and localization patterns between patient and control samples. Pay particular attention to immune tissues, liver samples, and cell types affected in the immunodeficiency phenotype.

Second, investigate V-ATPase complex assembly and function. Since ATP6AP1 (Ac45) is critical for V-ATPase assembly and function, use co-immunoprecipitation with ATP6AP1 antibodies to assess its interaction with other V-ATPase components in patient-derived cells. This can reveal whether pathogenic mutations affect complex formation or stability.

Third, examine protein glycosylation patterns. Research has identified abnormal protein glycosylation in patients with hemizygous missense mutations in ATP6AP1 . Combine ATP6AP1 immunodetection with glycosylation analyses to determine if altered ATP6AP1 function affects protein processing pathways.

Fourth, assess tissue-specific effects. Different Ac45 protein isoforms have been identified in human brain, liver, and B cells, indicating tissue-specific regulation of organelle acidification . Use isoform-specific ATP6AP1 antibodies to investigate how mutations affect these tissue-specific patterns and correlate with clinical manifestations.

Finally, conduct functional rescue experiments. In studies where yeast V-ATPase assembly factor Voa1 was complemented with the processed C-terminal domain of Ac45 , similar approaches can be used to test the functional consequences of patient-derived ATP6AP1 mutations and potential therapeutic interventions.

How can researchers evaluate the specificity of ATP6AP1 antibodies in their experimental system?

Establishing antibody specificity is critical for reliable results. For ATP6AP1 antibodies, implement these validation strategies:

First, perform Western blotting with positive and negative controls. Use tissues or cell lines known to express ATP6AP1 (positive control) and those with low or no expression (negative control). Observe whether the antibody detects a single band at the expected molecular weight (approximately 95 kDa in Arabidopsis thaliana) . For human samples, verify the appropriate molecular weight based on the specific isoform under investigation.

Second, conduct knockdown/knockout validation. Compare ATP6AP1 detection in wild-type cells versus those with siRNA knockdown or CRISPR-Cas9 knockout of ATP6AP1. A specific antibody will show significantly reduced or absent signal in knockdown/knockout samples.

Third, perform peptide competition assays. Pre-incubate the ATP6AP1 antibody with excess immunizing peptide before application to your samples. Specific binding should be blocked, while non-specific binding will remain.

Fourth, evaluate cross-reactivity. Test the antibody against related proteins or in samples from different species. For example, some ATP6AP1 antibodies have been tested for cross-reactivity with Aspergillus niger and found to be non-reactive , confirming specificity.

Fifth, compare results from multiple antibodies. Use different ATP6AP1 antibodies that target distinct epitopes. Agreement between results from different antibodies increases confidence in specificity. Consider using both monoclonal (e.g., clone 85.1) and polyclonal options for validation .

Finally, consider mass spectrometry validation. For definitive confirmation, immunoprecipitate ATP6AP1 using your antibody and analyze the precipitated proteins by mass spectrometry to verify that ATP6AP1 is indeed the captured protein.

What approaches are recommended for investigating ATP6AP1's role in cancer-associated fibroblast (CAF) interactions?

ATP6AP1's positive correlation with cancer-associated fibroblasts (CAFs) in colorectal cancer suggests important functional interactions that can be investigated using these methodological approaches:

First, perform dual immunofluorescence staining using ATP6AP1 antibodies combined with established CAF markers. Research shows that ATP6AP1 expression positively correlates with CAF markers such as ACAT2, S100A4, VIM, and PDGFRA/B in both colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) . Co-localization analysis can reveal spatial relationships between ATP6AP1-expressing cells and CAFs within the tumor microenvironment.

Second, establish co-culture systems combining cancer cells with CAFs that have different ATP6AP1 expression levels (natural variation or engineered). Measure changes in CAF activation markers, extracellular matrix production, and cancer cell invasiveness to determine functional consequences of ATP6AP1 modulation.

Third, perform secretome analysis of conditioned media from these co-culture systems. Compare the profile of secreted factors between conditions with normal versus altered ATP6AP1 expression to identify potential signaling mechanisms.

Fourth, investigate mechanistic pathways using phosphoproteomic analysis. After immunoprecipitation with ATP6AP1 antibodies, analyze the phosphorylation status of associated proteins to identify ATP6AP1-dependent signaling networks in CAF-cancer cell interactions.

Finally, conduct in vivo studies using patient-derived xenografts with ATP6AP1 manipulation in either cancer cells or stromal components. This approach can validate in vitro findings and assess the therapeutic potential of targeting ATP6AP1 in the context of tumor-stromal interactions.

What are common challenges when using ATP6AP1 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with ATP6AP1 antibodies. Here are the most common issues and recommended solutions:

Challenge 1: Weak or No Signal in Western Blots

• Solution: Optimize protein extraction methods for membrane proteins. Use RIPA buffer with protease inhibitors and avoid repeated freeze-thaw cycles.

• Increase antibody concentration or extend incubation time to overnight at 4°C.

• Consider using enhanced chemiluminescence (ECL) substrates with higher sensitivity.

• Try alternative antibodies that target different epitopes of ATP6AP1.

Challenge 2: High Background in Immunohistochemistry/Immunofluorescence

• Solution: Increase blocking time (2-3 hours) with 5-10% normal serum from the same species as the secondary antibody.

• Optimize antibody dilutions; start with manufacturer recommendations and adjust as needed.

• Include additional washing steps with 0.1% Tween-20 in PBS.

• Pre-absorb secondary antibodies with tissue powder from the species being examined.

Challenge 3: Cross-Reactivity with Unrelated Proteins

• Solution: Validate antibody specificity using knockout/knockdown controls.

• Use peptide competition assays to confirm binding specificity.

• Try monoclonal antibodies like clone 85.1 for increased specificity .

• Include appropriate negative controls (secondary antibody alone, isotype controls).

Challenge 4: Inconsistent Results Between Applications

• Solution: Different applications may require different antibody clones. An antibody that works well for Western blotting may not be optimal for IHC due to epitope accessibility.

• Verify that the antibody has been validated for your specific application.

• Consider the nature of your sample preparation (native vs. denatured proteins).

Challenge 5: Detection of Multiple Bands

• Solution: This could indicate detection of different isoforms, post-translational modifications, or degradation products. Use tissue-specific positive controls, as different Ac45 protein isoforms have been identified in human brain, liver, and B cells .

• Include protease inhibitors during sample preparation to minimize degradation.

• Verify bands with additional antibodies targeting different epitopes.

What are the optimal storage and handling conditions for maintaining ATP6AP1 antibody performance?

Proper storage and handling of ATP6AP1 antibodies is crucial for maintaining their performance and extending their usable lifespan:

For long-term storage, most ATP6AP1 antibodies should be stored at -20°C in small aliquots to minimize freeze-thaw cycles. Repeated freezing and thawing can lead to antibody degradation and loss of activity. If provided in liquid form (typically at 100 μg/ml concentration), create working aliquots of 10-20 μl upon receipt .

When handling, always wear gloves to prevent contamination with skin proteins or proteases. Centrifuge the antibody vial briefly before opening to collect all liquid at the bottom of the vial, especially after thawing from -20°C storage.

For working dilutions, prepare them fresh on the day of use using appropriate diluents. For immunohistochemistry applications, antibody diluent with background-reducing components is recommended. For Western blotting, dilute in 5% non-fat milk or BSA in TBST.

Monitor antibody performance over time by including consistent positive controls with each experiment. A decrease in signal intensity or increase in background may indicate antibody degradation.

If using conjugated ATP6AP1 antibodies (e.g., HRP, PE, FITC, or Alexa Fluor® conjugates), protect them from light during handling and storage to prevent photobleaching of the fluorophores .

How might ATP6AP1 antibodies contribute to understanding the mechanism of ATP6AP1 in immunotherapy response?

Recent research has revealed significant correlations between ATP6AP1 expression and various immune checkpoints in colorectal cancer, suggesting potential roles in immunotherapy response . To investigate this mechanistic connection, researchers can employ ATP6AP1 antibodies in these innovative approaches:

First, perform multiplex immunohistochemistry/immunofluorescence to simultaneously detect ATP6AP1 and immune checkpoint molecules (PDCD1, CD276, VTCN1, etc.) in patient samples. This will enable spatial relationship analysis between ATP6AP1 expression and immune checkpoint distribution in the tumor microenvironment, providing insights into their potential functional interaction.

Second, conduct immunoprecipitation-mass spectrometry (IP-MS) using ATP6AP1 antibodies to identify novel protein-protein interactions between ATP6AP1 and components of immune signaling pathways. This unbiased approach may reveal previously unknown mechanisms by which ATP6AP1 influences immune responses.

Third, develop ATP6AP1 expression modulation models (overexpression/knockdown) in tumor and immune cells to assess the impact on immune checkpoint expression and function. Combined with flow cytometry using ATP6AP1 antibodies, researchers can quantify how ATP6AP1 levels affect immune cell phenotypes and activation states.

Fourth, integrate ATP6AP1 antibody-based tissue analysis with patient immunotherapy response data. By correlating ATP6AP1 expression patterns with clinical outcomes in patients receiving immune checkpoint inhibitors, researchers can evaluate its potential as a predictive biomarker for immunotherapy response.

Finally, investigate the relationship between ATP6AP1's role in V-ATPase function and antigen presentation pathways. Since V-ATPase activity affects endosomal/lysosomal pH, which is crucial for antigen processing, ATP6AP1 antibodies could help elucidate how alterations in this protein impact the cancer-immunity cycle.