ANXA11 Antibody

What is the functional significance of ANXA11 in cellular pathways?

ANXA11 functions as a calcium-binding protein with diverse roles in cellular processes. Its N-terminus contains a low-complexity domain that mediates phase transitions required for condensation into membraneless RNA granules, while its C-terminus harbors four calcium-binding annexin domains enabling regulated interactions with negatively-charged lysosomal membranes . ANXA11 plays a critical role in the early secretory pathway by physically associating with Sec31A through the adaptor function of ALG-2, maintaining architectural and functional features of the endoplasmic reticulum exit sites (ERES) .

For effective investigation of ANXA11 function, researchers should consider both its phase separation properties and membrane-binding capabilities in experimental designs, particularly when studying its role in RNA-lysosome tethering mechanisms disrupted in neurodegenerative conditions.

What methodological approaches are recommended for detecting ANXA11 in tissue samples?

For optimal detection of ANXA11 in tissue samples, immunohistochemistry protocols with antigen retrieval in 0.01 M citrate buffer (pH 6.0) have proven effective. Recommended primary antibody dilution ranges from 1:400 (Proteintech, 10,479–2-A) for paraffin-embedded sections with overnight incubation at 4°C . For fluorescent detection, Alexa Fluor-conjugated secondary antibodies yield optimal results when visualized with confocal microscopy.

When analyzing co-localization with other proteins (e.g., TDP-43), researchers should perform z-stack imaging with subsequent maximum-intensity projection to capture the entire thickness of the section. Region of interest (ROI) creation around specific anatomical structures (such as ventral horns in spinal cord) allows for standardized quantification across specimens .

How should researchers validate ANXA11 antibody specificity?

Validation of ANXA11 antibody specificity requires a multi-faceted approach:

• Western blot analysis to confirm recognition of the expected 54.4 kDa protein (noting that two reported isoforms exist)

• Comparison of staining patterns across multiple antibodies targeting different epitopes

• Knockdown/knockout controls using siRNA or CRISPR-Cas9 to demonstrate specificity

• Pre-absorption controls with recombinant ANXA11 protein

• Cross-validation in multiple species when studying orthologues (ANXA11 variants exist in canine, porcine, monkey, mouse and rat models)

When working with mutant forms of ANXA11, researchers should confirm antibody recognition of the variant protein, as mutations may alter epitope accessibility or protein conformation.

What experimental approaches are recommended for studying ANXA11 mutation effects on protein aggregation?

Investigating ANXA11 mutation effects on protein aggregation requires an integrated methodology:

• In vitro phase separation assays: Using recombinant ANXA11 proteins (wild-type and mutant variants) to assess abnormal phase separation properties under controlled conditions. This approach has successfully demonstrated that the p.Asp40Ile variant is more aggregation-prone than the ALS-associated p.Asp40Gly variant .

• Fibroblast stress granule dynamics: Patient-derived fibroblasts can reveal defects in stress granule formation, dynamics, and clearance. Time-lapse imaging following stress induction allows quantification of assembly/disassembly kinetics .

• Muscle histopathology: Analysis of patient muscle biopsies using super-resolution imaging can reveal characteristic aggregation patterns, such as the pearl strips or large complex structures in the sarcoplasm and layered subsarcolemmal chains observed with the p.Asp40Ile variant .

• Biochemical fractionation: Separation of protein samples into TBS-soluble, sarkosyl-soluble, and sarkosyl-insoluble fractions, with subsequent Western blotting, provides quantitative assessment of aggregation status. Elevated monomeric protein in the sarkosyl-insoluble fraction indicates increased aggregation .

How can researchers effectively investigate ANXA11-TDP-43 interactions in disease models?

To investigate ANXA11-TDP-43 interactions:

• Co-immunoprecipitation: Pulldown of TDP-43 and its interacting proteins can confirm direct interaction with ANXA11 in pathological states .

• Co-localization analysis: Confocal microscopy with antibodies against ANXA11, TDP-43, and p62 can reveal spatial relationships between these proteins. In ANXA11-mutant models, TDP-43 forms cytoplasmic inclusions co-aggregating with mutant ANXA11 around the nucleus .

• Temporal progression studies: Longitudinal analysis of disease models can track the evolution of TDP-43 mislocalization. In ANXA11-mutant mice, initial cytoplasmic TDP-43 inclusions at 2 months progress to almost complete cytoplasmic translocation by 9 months .

• Functional assays: Assessment of downstream effects of TDP-43 dysregulation, such as cryptic exon formation in iPSC-derived neurons expressing mutant ANXA11, provides functional evidence of pathological interactions .

What strategies are effective for investigating ANXA11's role in the early secretory pathway?

To investigate ANXA11's role in the early secretory pathway:

• siRNA-mediated knockdown: Depletion of ANXA11 or ALG-2 can reveal their impact on Sec31A association with ERES and ERES positioning within cells .

• Live-cell imaging: Tracking the population of Sec31A that is stably associated with the ERES in control versus ANXA11-depleted cells provides dynamic information about ERES maintenance .

• ER-to-Golgi transport assays: Synchronous transport assays of transmembrane cargoes in ANXA11-knockdown cells have demonstrated accelerated transport, suggesting a regulatory role in secretory pathway function .

• Immunofluorescence analysis: Assessing ERES distribution can reveal whether ANXA11 depletion causes scattering of juxtanuclear ERES to the cell periphery, disrupting the architectural organization of the early secretory pathway .

How can multiomics approaches advance understanding of ANXA11 pathophysiology?

Multiomics strategies provide comprehensive insights into ANXA11 pathophysiology:

Studies of the ANXA11 P93S variant demonstrated that pathogenic signatures varied significantly between neurons and microglia, with microglia showing more pronounced dysregulation. This highlights the importance of investigating multiple cell types when studying ANXA11-related diseases .

How do researchers distinguish between different ANXA11-related pathologies in experimental models?

Different ANXA11 mutations produce distinct phenotypes that can be characterized through:

• Age of onset analysis: Mutations affecting the Asp40 residue can cause adult-onset ALS and multisystem proteinopathy, but the p.Asp40Ile variant leads to severe childhood-onset oculopharyngeal muscular dystrophy .

• Aggregation propensity assessment: Different mutations enhance aggregation to varying degrees, with p.Asp40Ile predicted to enhance aggregation propensity more than other changes affecting this residue .

• Tissue-specific pathology: Muscle histopathology reveals characteristic patterns depending on the mutation and disease phenotype. Super-resolution imaging can identify distinct aggregation patterns such as pearl strips or layered subsarcolemmal chains .

• TDP-43 pathology progression: The extent and temporal progression of TDP-43 mislocalization varies among ANXA11 mutations. Late-onset ALS with the P36R mutation shows characteristic cytoplasmic TDP-43 inclusions that worsen with disease progression .

What methodologies are recommended for detecting ANXA11 aggregates in patient-derived samples?

For detection of ANXA11 aggregates in patient samples:

• Biochemical fractionation: Sequential extraction with increasing detergent strength to separate soluble and insoluble protein fractions, followed by Western blotting .

• Super-resolution imaging: Required to visualize complex aggregate structures such as pearl strips in the sarcoplasm and layered subsarcolemmal chains .

• Co-localization studies: Multiple immunofluorescence labeling with antibodies against ANXA11, TDP-43, and p62 to characterize aggregate composition and identify potential degradation pathway involvement .

• Electron microscopy: Valuable for ultrastructural characterization of aggregates, particularly in muscle biopsies where filamentous or amorphous inclusions may be present .

How can researchers effectively model the impact of ANXA11 mutations on cellular stress response?

To model the impact of ANXA11 mutations on cellular stress response:

• Patient-derived fibroblast analysis: Studies have revealed defects in stress granule dynamics and clearance in fibroblasts from patients with ANXA11 mutations .

• iPSC-derived neuronal models: Allow examination of cell type-specific effects of ANXA11 mutations, including decreased colocalization of lysosomes and decreased neuritic RNA .

• Stress induction protocols: Application of various stressors (oxidative stress, heat shock, etc.) followed by time-course analysis of stress granule formation and resolution .

• RNA-lysosome tethering assays: Quantitative assessment of RNA granule-lysosome interactions in axons, as disease-associated mutations disrupt this tethering function .

What factors should researchers consider when selecting an ANXA11 antibody for specific applications?

When selecting an ANXA11 antibody:

• Application compatibility: Verify validated applications for each antibody (Western blot, immunoprecipitation, immunocytochemistry, flow cytometry) .

• Epitope location: Consider whether the antibody targets N-terminal (low-complexity domain) or C-terminal (annexin domains) regions, particularly important when studying mutations that may affect epitope accessibility.

• Species reactivity: Confirm cross-reactivity with the species being studied (human, mouse, rat, etc.) .

• Clone type: Monoclonal antibodies offer greater specificity but potentially limited epitope recognition; polyclonal antibodies provide broader epitope recognition but potential batch-to-batch variability.

• Conjugation requirements: Determine if direct fluorophore conjugation is needed for applications like flow cytometry or multiplex immunofluorescence.

What controls are essential when using ANXA11 antibodies in experimental settings?

Essential controls include:

• Positive control tissue/cell line: Samples known to express ANXA11 at detectable levels.

• Negative control (knockdown/knockout): Samples where ANXA11 expression has been reduced or eliminated.

• Isotype control: For immunohistochemistry/immunofluorescence to assess non-specific binding.

• Secondary antibody-only control: To evaluate background signal.

• Cross-reactivity assessment: Particularly important when studying multiple annexin family members simultaneously.

• Peptide competition: Pre-incubation of antibody with the immunizing peptide to confirm specificity.

What are the optimal conditions for ANXA11 visualization in tissue sections?

For optimal visualization in tissue sections:

• Fixation protocol: 4% paraformaldehyde after PBS perfusion, followed by paraffin embedding has proven effective .

• Antigen retrieval: Heat-induced epitope retrieval in 0.01 M citrate buffer (pH 6.0) .

• Primary antibody dilution: 1:400 for rabbit anti-ANXA11 (Proteintech, 10,479–2-A) with overnight incubation at 4°C .

• Detection method: Both chromogenic (ABC kit, Vector) and fluorescent (Alexa Fluor-conjugated secondary antibodies) approaches have been validated .

• Imaging parameters: For fluorescence, confocal microscopy with z-stack acquisition and maximum intensity projection provides optimal visualization of aggregate structures .

What technological advancements might enhance ANXA11 research methodologies?

Emerging technologies with potential to advance ANXA11 research include:

• Live-cell phase separation imaging: Real-time visualization of ANXA11 condensation and interactions with RNA granules and lysosomes.

• Cryo-electron microscopy: Structural characterization of wild-type versus mutant ANXA11 to understand conformational changes affecting function.

• Proximity labeling approaches: BioID or APEX2 fusion proteins to identify the ANXA11 interactome in different cellular compartments.

• Single-cell multiomics: Integrated analysis of transcriptome, proteome, and phenotypic changes in individual cells expressing wild-type or mutant ANXA11.

• In situ structural analysis: Techniques like STORM or PALM super-resolution microscopy to characterize aggregate structure and composition with nanometer precision.

What methodological approaches could better elucidate the relationship between ANXA11 and neuroinflammation?

Given the emerging connection between ANXA11 and immune dysfunction:

• Microglia-neuron co-culture systems: To investigate cell-cell interactions and inflammatory signaling in the context of ANXA11 mutations.

• Interferon signaling pathway analysis: Focused examination of this pathway highlighted in multiomics studies of ANXA11 P93S variant .

• In vivo neuroinflammation models: Using ANXA11-mutant mice to assess glial activation, cytokine production, and blood-brain barrier integrity.

• Single-nucleus RNA sequencing: To identify cell type-specific transcriptional changes in ANXA11-related pathologies.

• Spatial transcriptomics: To map inflammatory signatures in relation to ANXA11 aggregate distribution in tissue sections.