AQP1 Antibody

What is AQP1 and why are antibodies against it important for research?

AQP1 is a 28 kDa integral membrane protein that forms water-specific channels, facilitating water transport across cell membranes. It was originally identified in red blood cells and renal proximal tubules but is now known to be expressed in numerous tissues . AQP1 antibodies are essential research tools for studying water transport mechanisms, tissue-specific expression patterns, and pathological conditions involving fluid homeostasis disruptions.

The protein has a calculated molecular weight of 269 amino acids (29 kDa), though observed molecular weights in experimental conditions can range from 25-28 kDa and 35-50 kDa due to post-translational modifications like glycosylation .

What are the primary applications for AQP1 antibodies in research?

AQP1 antibodies are utilized across multiple experimental platforms:

These applications enable researchers to examine AQP1 expression, localization, and interactions in various experimental contexts .

In which tissues is AQP1 normally expressed?

AQP1 demonstrates a distinctive expression pattern across human tissues, with varying expression levels:

High expression levels:

• Choroid plexus

• Kidney (proximal tubules)

• Hepatobiliary ducts

• Gallbladder

Moderate expression levels:

• Hippocampus

• Ependymal cell layer of the central nervous system

• Lung and bronchial epithelium

• Urinary bladder

• Synovium and articular cartilage

• Breast epithelium

• Anal mucosa

Low expression levels:

• Lymphatic endothelium of the heart

• Epididymis

• Adrenal medulla

• Fetal membranes

• Various other CNS regions

Understanding this distribution pattern is crucial for experimental design and interpreting immunostaining results.

How can researchers distinguish between different glycosylated forms of AQP1 in experimental settings?

AQP1 undergoes post-translational modifications, particularly glycosylation, resulting in molecular weight variations from 25-28 kDa (non-glycosylated) to 35-50 kDa (glycosylated forms) . To distinguish these forms:

• Deglycosylation experiments: Treat samples with enzymes like PNGase F prior to Western blotting

• Gradient gel electrophoresis: Utilize 5-20% SDS-PAGE gels for better separation of different AQP1 forms

• Migration pattern analysis: Compare observed bands with predicted molecular weights

• Tissue-specific controls: Include positive controls from tissues known to express differently glycosylated AQP1 forms

For example, in Western blot analysis, researchers should run samples under reducing conditions (30 μg/lane) with appropriate kidney tissue lysates as positive controls, as demonstrated in validation studies using rat and mouse kidney tissues .

What are the critical considerations when studying AQP1 antibodies in neuromyelitis optica spectrum disorder (NMOSD) research?

When investigating AQP1 antibodies in NMOSD contexts, researchers should:

• Use live cell-based assays (CBAs): These provide higher sensitivity for detecting clinically relevant antibodies that target extracellular domains

• Avoid serum preabsorption with liver powder: This can lead to loss of AQP1 antibodies

• Include appropriate controls: Both AQP4-positive and AQP4-negative NMOSD samples, MS samples, and healthy controls

• Consider epitope specificity: Focus on methods that specifically identify antibodies targeting the extracellular domain of AQP1, which have potential pathogenic roles

• Apply standardized screening protocols: Dilute serum 1:20 and 1:40 in PBS/FCS for 1 hour at 4°C for optimal detection

Research indicates conflicting findings regarding AQP1 antibodies in NMOSD, with some studies reporting their presence while others finding no evidence, highlighting the importance of methodological consistency .

How do AQP1 antibodies perform in multi-tissue microarray (TMA) validation, and what does this reveal about tissue-specific detection challenges?

Multi-tissue microarray validation reveals important considerations for AQP1 antibody applications:

• Tissue-specific signal intensity varies: Antibody performance differs significantly across tissues due to varying expression levels and potential cross-reactivity

• Antigen retrieval methods affect detection: For IHC applications, tissues may require different antigen retrieval methods (TE buffer pH 9.0 versus citrate buffer pH 6.0)

• Background signal considerations: Endogenous peroxidase activity in certain tissues requires careful blocking and washing steps

• Antibody clone selection matters: Different clones may exhibit variable performance across tissue types based on epitope accessibility

Successful TMA validation enables researchers to confidently apply AQP1 antibodies across diverse experimental contexts while understanding tissue-specific limitations.

What are the optimal procedures for Western blot detection of AQP1?

For optimal Western blot detection of AQP1:

• Sample preparation:

  • Use 30 μg of protein lysate per lane

  • Run under reducing conditions

  • Include positive controls (rat/mouse kidney lysates)

• Gel electrophoresis:

  • Utilize 5-20% SDS-PAGE gradient gels

  • Run at 70V (stacking)/90V (resolving) for 2-3 hours

• Transfer conditions:

  • Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

• Blocking and antibody incubation:

  • Block with 5% non-fat milk/TBS for 1.5 hours at room temperature

  • Incubate with primary antibody at 0.5 μg/mL overnight at 4°C

  • Wash with TBS-0.1% Tween (3 times, 5 minutes each)

  • Incubate with secondary antibody (e.g., goat anti-rabbit IgG-HRP) at 1:5000 dilution for 1.5 hours at room temperature

• Signal development:

  • Develop using Enhanced Chemiluminescent detection kit

  • Expected bands: 25-28 kDa (non-glycosylated) and 35-50 kDa (glycosylated forms)

What protocol modifications are needed for successful immunohistochemistry (IHC) detection of AQP1?

For optimal IHC detection of AQP1:

• Sample preparation:

  • Paraffin-embedded sections

  • Heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• Blocking and antibody incubation:

  • Block with 10% goat serum

  • Incubate with primary antibody (2 μg/ml) overnight at 4°C

  • Use peroxidase-conjugated goat anti-rabbit IgG as secondary antibody (30 minutes at 37°C)

• Signal development:

  • Develop using DAB as chromogen

  • Counterstain with hematoxylin for context

• Tissue-specific considerations:

  • For kidney tissue: Expect strong staining in proximal and distal convoluted tubules

  • For liver tissue: Hepatobiliary ducts will show strong positivity

  • For brain tissue: Choroid plexus will show distinct staining pattern

AQP1 staining is typically present in both distal and proximal convoluted tubules in the renal cortex, with weaker staining in collecting ducts, while glomeruli remain negative .

What considerations are critical for flow cytometry applications with AQP1 antibodies?

For successful flow cytometry using AQP1 antibodies:

• Cell preparation:

  • Fix cells with 4% paraformaldehyde

  • Block with 10% normal goat serum

• Antibody incubation:

  • Incubate with primary antibody (1 μg/1×10^6 cells) for 30 minutes at 20°C

  • Use fluorophore-conjugated secondary antibody (e.g., DyLight®488 conjugated goat anti-rabbit IgG, 5-10 μg/1×10^6 cells) for 30 minutes at 20°C

• Controls:

  • Include isotype control antibody (e.g., rabbit IgG, 1 μg/1×10^6 cells)

  • Include unlabeled sample as additional control

• Analysis considerations:

  • Overlay histogram showing cell populations

  • Adjust compensation for multicolor analysis

  • Gate appropriately based on controls

Flow cytometry with AQP1 antibodies enables quantitative assessment of expression levels and can be particularly useful for detecting membrane-associated versus internalized AQP1.

How can researchers address background issues in AQP1 immunohistochemistry?

Background issues in AQP1 immunohistochemistry can be addressed through the following systematic approaches:

• Optimize blocking:

  • Increase blocking time to 2 hours

  • Try alternate blocking agents (BSA, normal serum from secondary antibody species)

  • Consider using commercial blocking solutions specifically designed for IHC

• Adjust antibody concentrations:

  • Titrate primary antibody (start with 1:12000 dilution for high-expressing tissues)

  • Reduce secondary antibody concentration

  • Increase washing duration and frequency (5× washes of 5 minutes each)

• Antigen retrieval modifications:

  • Test both TE buffer pH 9.0 and citrate buffer pH 6.0

  • Optimize retrieval time and temperature

  • Consider pressure cooker versus microwave methods

• Endogenous peroxidase quenching:

  • Treat sections with 0.3% H₂O₂ in methanol for 30 minutes

  • For tissues with high endogenous peroxidase activity, consider fluorescent detection instead

What are the primary explanations for unexpected molecular weight variations in AQP1 Western blot analysis?

When facing unexpected molecular weight variations in AQP1 Western blots:

• Post-translational modifications:

  • Glycosylation causes higher molecular weight bands (35-50 kDa)

  • Phosphorylation may alter migration patterns

  • Ubiquitination can produce higher molecular weight species

• Protein aggregation:

  • Insufficient sample denaturation

  • Boiling time optimization (too long can cause aggregation)

  • Try alternative reducing agents (DTT vs. β-mercaptoethanol)

• Proteolytic degradation:

  • Add additional protease inhibitors to lysis buffer

  • Maintain cold chain throughout sample preparation

  • Avoid repeated freeze-thaw cycles

• Tissue-specific variations:

  • Different tissues express variant forms of AQP1

  • Species-specific differences in post-translational modifications

  • Developmental stage-specific expression patterns

Expected molecular weights include 25-28 kDa (non-glycosylated form) and 35-50 kDa (glycosylated forms), though this can vary by tissue type and experimental conditions.

What strategies can be employed when AQP1 antibodies fail to detect the protein in tissues known to express it?

When facing detection failures in tissues known to express AQP1:

• Epitope accessibility:

  • Try multiple antibodies targeting different epitopes

  • Consider antibodies against both N-terminal and C-terminal regions

  • For membrane proteins, detergent permeabilization may be required

• Fixation effects:

  • Different fixatives can mask epitopes

  • For formalin-fixed tissues, extend antigen retrieval time

  • Consider dual antigen retrieval methods (heat + enzymatic)

• Expression level considerations:

  • Increase antibody concentration for low-expressing tissues

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use signal amplification systems (TSA, polymeric detection)

• Sample handling:

  • Protein degradation during processing

  • Optimize tissue preservation protocols

  • Consider fresh frozen samples for difficult epitopes

How can researchers effectively use AQP1 antibodies to study its potential role in CO₂ transport and metabolism?

Recent research suggests AQP1 may function as part of a CO₂ metabolon, linking facilitated diffusion across membranes with anion exchange and interconversion of dissolved CO₂ and carbonic acid in the cytosol . To investigate this:

• Co-immunoprecipitation studies:

  • Use AQP1 antibodies (0.5-4.0 μg for 1.0-3.0 mg protein lysate) to pull down protein complexes

  • Probe for interacting partners (e.g., ankyrin-1, carbonic anhydrase)

  • Analyze complex formation under various physiological conditions

• Functional assays:

  • Combine AQP1 immunodetection with pH-sensitive dyes

  • Correlate AQP1 expression with CO₂ transport rates

  • Use selective inhibitors to distinguish AQP1-mediated from other transport mechanisms

• Subcellular localization studies:

  • Employ super-resolution microscopy with AQP1 antibodies

  • Use co-localization studies with known CO₂ metabolon components

  • Analyze redistribution following physiological stimuli

What are the methodological considerations for detecting AQP1 autoantibodies in clinical samples?

For detecting AQP1 autoantibodies in clinical samples:

• Cell-based assay (CBA) approach:

  • Use transiently transfected HEK293A cells expressing AQP1 fused C-terminally to emerald green fluorescence protein

  • Block cells with goat IgG in PBS/10% FCS

  • Incubate with patient serum diluted 1:20 and 1:40 in PBS/FCS for 1 hour at 4°C

  • Detect bound antibodies using Cy3™-conjugated secondary antibodies

• Critical methodological considerations:

  • Avoid serum preabsorption with liver powder (can lead to loss of AQP1 antibodies)

  • Use live cell assays rather than fixed cells for detecting pathologically relevant antibodies

  • Include appropriate positive and negative controls

  • Validate results using multiple detection methods

• Interpretation challenges:

  • Distinguish between antibodies targeting extracellular versus intracellular domains

  • Consider cross-reactivity with other aquaporins

  • Correlate antibody titers with clinical manifestations

How do different AQP1 antibody clones compare in their ability to detect the protein across diverse experimental platforms?

Different AQP1 antibody clones show varying performance across experimental platforms:

For critical applications:

• Western blotting: Polyclonal antibodies often provide higher sensitivity

• IHC: Monoclonal antibodies may offer better specificity and lower background

• Quantitative applications: Recombinant monoclonal antibodies provide consistent results across experiments

When selecting an antibody clone, researchers should consider the specific application, target species, and whether detection of specific post-translational modifications is important for their research question.