AQP12A/AQP12B Antibody

What are the fundamental characteristics of AQP12A and AQP12B proteins?

AQP12A and AQP12B represent the most recently identified members of the mammalian aquaporin (AQP) family. Human AQP12 has a canonical amino acid length of 295 residues and a protein mass of approximately 31.5 kilodaltons. Unlike other aquaporins that are distributed across multiple tissues, AQP12 shows remarkable tissue specificity, being predominantly expressed in pancreatic acinar cells. In vitro expression studies have revealed that AQP12 is localized at intracellular sites rather than plasma membranes. Both proteins belong to the MIP/aquaporin (TC 1.A.8) protein family, which regulates water transport, lipid metabolism, and glycolysis in cells .

How do AQP12A and AQP12B differ from other aquaporin family members?

AQP12A and AQP12B are distinctive among the 13 identified human aquaporins (AQP0-AQP12) in several key aspects. While most aquaporins are integrated into plasma membranes for controlling water flow between cells and their environment, AQP12 proteins are primarily found in intracellular locations. Furthermore, whereas other AQPs are expressed across multiple tissue types, AQP12 expression appears to be specifically restricted to pancreatic tissue, suggesting a specialized function. Notably, knockout studies in mice have revealed that AQP12 deficiency does not cause obvious phenotypic changes under normal conditions, but significantly increases susceptibility to pancreatitis when challenged with a cholecystokinin-8 analog, indicating its potential role in pancreatic stress responses .

What are the critical considerations when selecting an AQP12A/AQP12B antibody for research applications?

When selecting an AQP12A/AQP12B antibody, researchers should consider several technical parameters. First, examine the specificity—whether the antibody targets AQP12A, AQP12B, or both. For instance, antibodies like ABIN655768 target specific amino acid regions (AA 236-264) from the C-terminal region of human AQP12B . Second, verify the host species and clonality; most available antibodies are rabbit polyclonal, which provides broader epitope recognition but may have batch-to-batch variation. Third, confirm the validated applications (Western Blot, ELISA, Flow Cytometry, Immunohistochemistry) with their recommended dilutions (typically WB: 1:500-1:2000, ELISA: 1:40000) . Finally, evaluate conjugation options (unconjugated, APC, FITC, Biotin, PE, HRP) based on your experimental design and detection systems .

How can researchers validate the specificity of AQP12A/AQP12B antibodies in their experimental systems?

Validating antibody specificity for AQP12A/AQP12B requires a multi-faceted approach. Begin with positive and negative tissue controls—pancreatic acinar cells should show expression while most other tissues should be negative. Implement knockout validation using AQP12-KO mouse samples or cell lines with CRISPR-Cas9 edited AQP12 genes as definitive negative controls. Perform peptide competition assays by pre-incubating the antibody with the immunogenic peptide (typically from the C-terminal region of human AQP12) before application to samples; this should abolish specific signals. Additionally, employ multiple antibodies targeting different epitopes of AQP12A/AQP12B to confirm staining patterns. Western blotting should reveal bands at approximately 31.5 kDa corresponding to the predicted molecular weight of AQP12 proteins. Finally, consider cross-reactivity testing with other aquaporin family members, particularly those with sequence homology to AQP12 .

What are the optimal conditions for Western blot analysis of AQP12A/AQP12B?

For optimal Western blot detection of AQP12A/AQP12B, sample preparation is critical. Extract proteins using buffers containing 1% SDS or other strong detergents to solubilize these membrane-associated proteins. Maintain samples at 4°C with protease inhibitors to prevent degradation. For electrophoresis, use 10-12% SDS-PAGE gels and transfer proteins to PVDF membranes (preferred over nitrocellulose for hydrophobic proteins). Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature. Apply primary AQP12A/AQP12B antibody at dilutions between 1:500-1:2000 (as specified for products like CSB-PA000912) and incubate overnight at 4°C . For detection, HRP-conjugated secondary antibodies at 1:5000-1:10000 dilutions are suitable, followed by ECL detection. When interpreting results, expect bands at approximately 31.5 kDa, though post-translational modifications may alter migration patterns. Jurkat cells have been documented as a positive control for Western blot analysis of AQP12 expression .

What methodological approaches are recommended for immunohistochemical detection of AQP12A/AQP12B in tissue samples?

For immunohistochemical detection of AQP12A/AQP12B in tissue samples, optimize fixation with 4% paraformaldehyde rather than formalin to preserve epitope accessibility. For paraffin-embedded sections, implement heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) to unmask antigens. When using antibodies like ABIN655768, apply at dilutions between 1:10-1:50 for IHC-P applications . For fluorescent detection, secondary antibodies conjugated with fluorophores like Alexa Fluor are preferable due to their brightness and photostability. Include appropriate blocking with 5-10% normal serum from the secondary antibody host species to minimize background. Due to AQP12's intracellular localization, permeabilization with 0.1-0.5% Triton X-100 is essential. When evaluating staining, pancreatic acinar cells should show positive cytoplasmic staining with potential concentration in specific intracellular compartments, while other tissues typically serve as negative controls .

How can AQP12A/AQP12B antibodies be utilized to investigate potential roles in pathological conditions beyond pancreatitis?

Recent research has identified altered expression patterns of aquaporins, including AQP12, in various pathological conditions, particularly in cancer. To investigate these roles, researchers can employ a multi-modal approach using AQP12A/AQP12B antibodies. First, perform comparative immunohistochemistry analysis on tissue microarrays containing normal and pathological samples from multiple organs, as demonstrated in clear cell renal cell carcinoma (ccRCC) studies . For quantitative assessment, implement digital pathology analysis with antibodies targeting both AQP12A and AQP12B to assess expression differences, using classification criteria based on the fraction of stained cells (0%, <25%, 25%-75%, >75%). Combine with transcriptomic analysis to correlate protein expression with mRNA levels across different pathological stages. Additionally, co-immunoprecipitation experiments using AQP12A/AQP12B antibodies can identify novel interaction partners in disease states. Finally, cell culture models with induced pathological conditions can be analyzed for changes in AQP12 expression and subcellular localization, providing insights into functional roles during disease progression .

What are the considerations for using AQP12A/AQP12B antibodies in flow cytometry applications?

When utilizing AQP12A/AQP12B antibodies for flow cytometry, several technical considerations are critical for successful analysis. First, since AQP12 proteins are predominantly intracellular, effective permeabilization is essential—use 0.1% saponin or 0.1-0.3% Triton X-100 after fixation with 2-4% paraformaldehyde. Select antibodies specifically validated for flow cytometry applications, such as those documented in catalog ABIN655768, which recommends dilutions around 1:10-1:50 for FACS applications . For multi-parameter analysis, choose antibody conjugates (FITC, PE, APC) that fit within your panel design to avoid fluorescence spillover. Include appropriate compensation controls and FMO (Fluorescence Minus One) controls to accurately set gates. When analyzing pancreatic cell suspensions or cell lines, consider using additional markers such as amylase to specifically identify acinar cells. Finally, implement proper gating strategies to exclude cell debris and doublets before analyzing AQP12A/AQP12B expression patterns .

What are common challenges encountered when using AQP12A/AQP12B antibodies, and how can researchers overcome them?

Researchers working with AQP12A/AQP12B antibodies frequently encounter several technical challenges. First, the high sequence homology between AQP12A and AQP12B (as evidenced by antibodies like OSA00153W-100UL that recognize both proteins) can make specific detection difficult . To address this, utilize antibodies raised against unique peptide sequences where the proteins differ, or validate with genetic knockout models specific to each variant. Second, AQP12's predominantly intracellular localization requires optimized permeabilization protocols—test increasing concentrations of detergents (0.1-0.5% Triton X-100) to improve access while maintaining epitope integrity. Third, low endogenous expression levels in most tissues except pancreas necessitates sensitive detection methods; consider signal amplification approaches like tyramine signal amplification for immunohistochemistry or highly sensitive ECL substrates for Western blotting. Fourth, high background in immunostaining can be reduced by extending blocking times (2-3 hours) with 5% BSA or normal serum. Finally, protein degradation during extraction can be minimized by working rapidly at 4°C with comprehensive protease inhibitor cocktails containing both serine and cysteine protease inhibitors .

How should researchers interpret contradictory AQP12A/AQP12B expression data between different detection methods?

When confronted with contradictory AQP12A/AQP12B expression data between different detection methods, researchers should implement a systematic analytical approach. First, recognize that discrepancies between mRNA and protein levels are biologically plausible due to post-transcriptional regulation mechanisms. For instance, analysis of ccRCC samples revealed inconsistencies between AQP12B mRNA upregulation in UALCAN database analysis and undetectable protein expression in immunohistochemistry . To resolve such contradictions, employ multiple antibodies targeting different epitopes of AQP12A/AQP12B proteins. Quantify protein expression using absolute quantification methods like selected reaction monitoring (SRM) mass spectrometry as an antibody-independent approach. Validate antibody specificity in knockout/knockdown systems prior to interpreting expression data. Consider tissue-specific post-translational modifications that might affect epitope accessibility or antibody binding. Examine subcellular fractionation coupled with Western blotting to determine if compartmentalization affects detection. Finally, validate findings across multiple biological replicates and independent cohorts, complementing antibody-based methods with orthogonal techniques like in situ hybridization for mRNA localization .

How can AQP12A/AQP12B antibodies complement genetic analysis approaches in understanding functional roles?

Integrating AQP12A/AQP12B antibody-based approaches with genetic analysis creates a powerful methodology for functional characterization. Begin with immunophenotyping of genetic variants identified through sequencing studies, such as the common nonsense variant in AQP12B (p.S152Tfs*24) found in chronic pancreatitis research . Use epitope-specific antibodies to determine if truncated proteins are expressed and stable. For variants with altered expression, implement immunolocalization studies to determine if subcellular distribution is affected, particularly important given AQP12's intracellular localization pattern. Combine with proximity ligation assays using AQP12A/AQP12B antibodies to assess if genetic variants affect protein-protein interactions. For functional studies, correlate antibody-based quantification of AQP12 expression levels with phenotypic readouts in genetic models, similar to the increased susceptibility to pancreatitis observed in AQP12-KO mice . Finally, implement ChIP-seq using antibodies against transcription factors predicted to regulate AQP12A/AQP12B expression to understand genetic regulation mechanisms. This integrated approach provides deeper insights than either method alone, connecting genotype to protein expression patterns and ultimately to functional consequences .

What experimental designs can determine if the nonsense variant in AQP12B (p.S152Tfs*24) produces a functional or non-functional protein?

To determine if the AQP12B nonsense variant (p.S152Tfs24) produces a functional or non-functional protein, implement a multi-faceted experimental design. First, generate constructs expressing wild-type AQP12B and the p.S152Tfs24 variant with epitope tags at both N-terminus and C-terminus. Transfect these into appropriate cell lines and perform Western blotting with antibodies recognizing the N-terminal tag to determine if the truncated protein is stable and expressed. Use AQP12B antibodies targeting epitopes upstream of the truncation site (residue 152) to confirm expression of the truncated protein. For functional analysis, measure water/solute transport capabilities in transfected cells using fluorescent probes or cell swelling assays. Conduct subcellular fractionation followed by immunoblotting to determine if the truncated protein maintains proper intracellular localization. Implement a complementation assay in AQP12-knockout pancreatic acinar cells challenged with CCK-8 to assess if the variant can rescue the heightened sensitivity to pancreatitis. Finally, perform co-immunoprecipitation experiments with known or predicted AQP12B interaction partners to determine if the truncated protein maintains its protein-protein interaction capabilities. This comprehensive approach will determine both the expression and functionality of this common variant protein .

What is the significance of AQP12A/AQP12B in non-cancerous pathological conditions and how can researchers effectively study this?

While AQP12A/AQP12B have been primarily studied in relation to pancreatic function, emerging evidence suggests potential roles in non-cancerous pathological conditions beyond pancreatitis. To investigate these roles, researchers should first perform comprehensive tissue expression profiling using immunohistochemistry with validated AQP12A/AQP12B antibodies across tissues affected by various inflammatory, degenerative, and metabolic disorders. For instance, chronic pancreatitis studies have shown genetic variants in AQP12A/AQP12B, though without significant association with disease predisposition . Design case-control studies measuring AQP12A/AQP12B protein levels in affected tissues and biological fluids, potentially identifying them as disease biomarkers. Utilize conditional knockout mouse models with tissue-specific deletion of AQP12A/AQP12B to assess vulnerability to disease induction in various organ systems. Implement time-course studies during disease progression to determine if expression changes precede or follow pathological alterations. For mechanistic understanding, perform co-immunoprecipitation with AQP12A/AQP12B antibodies followed by mass spectrometry to identify interaction partners that may change during disease states. Additionally, study epigenetic regulation of AQP12A/AQP12B expression during disease progression using ChIP-seq for histone modifications and DNA methylation analysis .

How can researchers investigate potential correlations between AQP12A/AQP12B expression and immune cell infiltration in tissue samples?

Investigating correlations between AQP12A/AQP12B expression and immune cell infiltration requires sophisticated methodological approaches combining immunostaining and computational analysis. Begin with multiplex immunofluorescence using AQP12A/AQP12B antibodies alongside markers for key immune cell populations (CD8+ T cells, CD4+ T cells, B cells, macrophages, neutrophils, and dendritic cells). Digital pathology analysis should quantify spatial relationships between AQP12-expressing cells and immune infiltrates, measuring distances and contact frequencies. Complement tissue studies with computational analyses similar to those implemented through the TIMER database for other aquaporins in ccRCC, which revealed significant correlations between certain AQPs and immune cell infiltration . For mechanistic insights, implement laser capture microdissection of AQP12-high versus AQP12-low regions followed by transcriptomic analysis of immune signatures. In experimental models, use flow cytometry with AQP12A/AQP12B antibodies combined with immune markers to quantify associations at single-cell resolution. Finally, in vitro co-culture systems with AQP12-expressing cells and various immune populations can help determine if these proteins influence immune cell recruitment, activation, or function through direct or indirect mechanisms .

What experimental approaches can determine if AQP12A/AQP12B proteins have immunomodulatory functions?

To determine potential immunomodulatory functions of AQP12A/AQP12B proteins, implement a comprehensive experimental framework spanning in vitro and in vivo systems. First, develop cell models with controlled expression of AQP12A/AQP12B (overexpression, knockdown, and knockout) in relevant cell types, particularly pancreatic acinar cells. Co-culture these modified cells with immune cell populations (T cells, B cells, macrophages) and measure changes in immune activation markers, cytokine production, and proliferation rates. Analyze secretomes from AQP12A/AQP12B-expressing cells to identify immunomodulatory factors using cytokine arrays and mass spectrometry. In animal models, compare immune responses to inflammatory challenges between wild-type and AQP12-knockout mice, particularly focusing on pancreatic inflammation models where AQP12-KO mice have shown increased susceptibility to pancreatitis . Implement adoptive transfer experiments with labeled immune cells to assess recruitment to tissues with varying AQP12A/AQP12B expression. For human studies, analyze correlations between AQP12A/AQP12B expression and immune markers in tissue samples from inflammatory conditions, potentially using approaches similar to those employed by TIMER analysis for other aquaporins in cancer . Finally, investigate if AQP12A/AQP12B antibodies themselves can modulate immune responses when applied to cell cultures or in passive immunization experiments .

How can AQP12A/AQP12B antibodies be utilized in subcellular localization studies to understand trafficking mechanisms?

Investigating AQP12A/AQP12B subcellular localization and trafficking mechanisms requires sophisticated imaging approaches combined with biochemical techniques. Begin with super-resolution microscopy (STED, STORM, or PALM) using AQP12A/AQP12B antibodies to achieve nanometer-scale resolution of intracellular distribution, critical for determining precise organelle associations of these predominantly intracellular proteins . Implement live-cell imaging using split-GFP complementation systems where one fragment is fused to AQP12 and the other to organelle markers, allowing real-time visualization of trafficking. For quantitative assessment of dynamic changes, perform pulse-chase experiments with photoconvertible protein-tagged AQP12 constructs. Complement imaging with biochemical fractionation studies using differential centrifugation followed by Western blotting with AQP12A/AQP12B antibodies to quantify distribution across subcellular compartments. To identify trafficking motifs, generate deletion and point mutation constructs of AQP12 and assess localization changes using immunofluorescence. Additionally, implement proximity labeling approaches like BioID or APEX2 fused to AQP12 to identify nearby proteins involved in trafficking machinery. Finally, examine trafficking responses to physiological stimuli relevant to pancreatic function, such as secretagogues or endoplasmic reticulum stress inducers, which may regulate AQP12 distribution during pancreatic acinar cell adaptation to various conditions .

What approaches can be used to study potential heterooligomerization between AQP12A and AQP12B, and with other aquaporin family members?

Investigating potential heterooligomerization between AQP12A, AQP12B, and other aquaporin family members requires multiple complementary approaches. First, implement Förster Resonance Energy Transfer (FRET) microscopy using differentially labeled AQP12A and AQP12B to detect close molecular associations (<10 nm) indicative of oligomerization. Complement this with Proximity Ligation Assays (PLA) using specific antibodies against AQP12A, AQP12B, and other aquaporins expressed in the same tissues to visualize protein interactions in situ with single-molecule sensitivity. For biochemical validation, perform co-immunoprecipitation experiments using AQP12A-specific antibodies followed by Western blotting for AQP12B and other aquaporins, and vice versa. Implement Blue Native PAGE to isolate and characterize native protein complexes containing AQP12A/AQP12B, followed by mass spectrometry for compositional analysis. Create chimeric constructs with split reporter proteins (BiFC) fused to potential interaction partners to visualize oligomerization in living cells. For functional assessment of heterooligomers, perform dominant-negative inhibition studies by co-expressing wild-type and mutant forms of AQP12A/AQP12B and measuring functional parameters. Finally, use computational modeling based on known aquaporin structures to predict interaction interfaces, followed by site-directed mutagenesis of key residues to validate their role in oligomerization. This multi-faceted approach will comprehensively characterize the oligomerization properties of these specialized aquaporins .

What emerging technologies could enhance AQP12A/AQP12B detection and functional characterization?

Several cutting-edge technologies show promise for advancing AQP12A/AQP12B research beyond current methodological limitations. Single-cell proteomics combined with spatial transcriptomics could reveal cell-type specific expression patterns and co-expression networks in tissues with heterogeneous cell populations. The development of nanobodies against AQP12A/AQP12B would overcome size limitations of conventional antibodies, enabling better access to intracellular epitopes and improved super-resolution imaging. CRISPR activation/interference (CRISPRa/CRISPRi) systems could provide precise temporal control of AQP12A/AQP12B expression for functional studies. Microfluidic organ-on-chip technologies incorporating pancreatic acinar cells with controlled AQP12A/AQP12B expression would allow physiological assessment under defined conditions. Mass cytometry (CyTOF) with metal-conjugated AQP12A/AQP12B antibodies could enable high-dimensional analysis of expression patterns alongside numerous other cellular markers. Cryo-electron microscopy could resolve the molecular structure of AQP12A/AQP12B, informing functional predictions and drug design. Synthetic biology approaches using engineered versions of AQP12A/AQP12B with introduced functionalities (light-sensitivity, ligand-gating) could enable precise control of activity. Finally, the application of artificial intelligence for image analysis of AQP12A/AQP12B immunostaining patterns could identify subtle expression changes and correlations with disease features not detectable by conventional means .