ACS11 Antibody

What is Annexin A11 (ANXA11) and what are its key functions in cellular processes?

Annexin A11 (ANXA11) is a member of the annexin family of calcium-dependent phospholipid-binding proteins that plays important roles in various cellular processes. Also known as ANX-A11, ANX11, or CAP50, it functions as a 56 kDa autoantigen and is identified as calcyclin-associated annexin 50 . ANXA11 is encoded by the gene ID 311 in humans with the accession number P50995 . The protein consists of amino acids from Met1 to Asp505 and participates in calcium-dependent vesicle trafficking, cell division, apoptosis, and calcium signaling pathways. ANXA11 has gained significant research interest due to its implications in autoimmune disorders and neurodegenerative diseases, making antibodies against this protein valuable research tools. The protein's structure includes calcium-binding domains that facilitate its interaction with membrane phospholipids, which is essential for its cellular functions.

What are the standard methods for validating ANXA11 antibodies before experimental use?

Validation of ANXA11 antibodies should follow a systematic approach to ensure specificity and reproducibility in research applications. A comprehensive validation protocol includes:

• Application-specific testing: ANXA11 antibodies should be validated for specific applications such as Western Blotting (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC), and Immunoprecipitation (IP) as indicated in the antibody specifications .

• Cross-reactivity assessment: Evaluate species reactivity, particularly when working with human samples. Commercial ANXA11 antibodies, such as CAU22689, are often validated specifically for human (Homo sapiens) ANXA11 .

• Positive and negative controls: Use cell lines or tissues known to express or lack ANXA11 to confirm specificity.

• Molecular weight verification: Confirm that the detected protein band corresponds to the expected molecular weight of ANXA11 (approximately 56 kDa).

• Antibody identity confirmation: For rigorous validation, consider using MALDI-TOF MS fingerprinting to confirm antibody identity, especially when obtaining antibodies from third parties . This method can circumvent lengthy denaturation, reduction, alkylation, and enzymatic digestion steps by using a simple formic acid hydrolysis approach that creates distinctive peptide fingerprints .

What are the recommended protocols for using ANXA11 antibodies in different experimental applications?

For optimal results across various experimental applications, researchers should follow these protocol guidelines:

Western Blotting (WB):

• Sample preparation: Lyse cells in a buffer containing protease inhibitors

• Protein quantification: Determine concentration via Bradford Assay

• SDS-PAGE: Load 10-20 μg protein per lane

• Transfer: Use PVDF membrane for optimal protein binding

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

• Primary antibody incubation: Dilute ANXA11 antibody (typically 1:1000) in blocking buffer and incubate overnight at 4°C

• Secondary antibody: Use HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

• Detection: Develop using ECL substrate and imaging system

Immunohistochemistry (IHC):

• Tissue preparation: Fix tissues in 10% neutral buffered formalin and embed in paraffin

• Sectioning: Cut 4-6 μm thick sections

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

• Blocking: 10% normal serum from the species of secondary antibody

• Primary antibody incubation: Apply diluted ANXA11 antibody and incubate overnight at 4°C

• Detection: Use appropriate detection system (e.g., ABC method) with DAB as chromogen

• Counterstaining: Hematoxylin for nuclear visualization

Immunocytochemistry (ICC):

• Cell fixation: 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: 0.2% Triton X-100 for 10 minutes

• Blocking: 5% normal serum for 1 hour

• Primary antibody: Incubate with diluted ANXA11 antibody overnight at 4°C

• Secondary antibody: Fluorophore-conjugated secondary antibody for 1 hour at room temperature

• Nuclear counterstain: DAPI or Hoechst

• Mounting: Anti-fade mounting medium

Immunoprecipitation (IP):

• Cell lysis: Use non-denaturing lysis buffer with protease inhibitors

• Pre-clearing: Incubate lysate with protein A/G beads

• Antibody binding: Add ANXA11 antibody to pre-cleared lysate and incubate overnight at 4°C

• Immunoprecipitation: Add protein A/G beads and incubate for 2-4 hours

• Washing: Wash beads 4-5 times with lysis buffer

• Elution: Boil beads in SDS sample buffer

• Analysis: Proceed with SDS-PAGE and Western blotting

How do polyclonal and monoclonal ANXA11 antibodies differ in research applications?

Polyclonal and monoclonal ANXA11 antibodies each offer distinct advantages and limitations for research applications:

When selecting between polyclonal and monoclonal antibodies for ANXA11 research, consider the experimental requirements, especially regarding specificity, signal strength, and the nature of the application. For instance, commercial polyclonal ANXA11 antibodies like CAU22689 are validated for WB, IHC, ICC, and IP applications , making them versatile research tools.

What controls should be included when performing immunoassays with ANXA11 antibodies?

Proper controls are essential for reliable ANXA11 antibody-based experiments. Researchers should implement the following controls:

• Positive tissue/cell controls: Include samples known to express ANXA11 at detectable levels to validate antibody performance.

• Negative tissue/cell controls: Use samples known to lack ANXA11 expression to confirm specificity.

• Primary antibody omission control: Perform the immunoassay without the primary ANXA11 antibody to identify non-specific binding of secondary antibodies.

• Isotype control: Use a non-specific antibody of the same isotype and concentration as the ANXA11 antibody to identify non-specific binding.

• Blocking peptide control: Pre-incubate the ANXA11 antibody with the immunizing peptide (Met1~Asp505 for antibodies like CAU22689 ) before application to confirm specificity.

• Concentration gradients: Test different antibody concentrations to determine optimal signal-to-noise ratios.

• Cross-reactivity controls: When studying ANXA11 in multiple species, include samples from each species to confirm reactivity.

• Loading controls: For Western blots, include housekeeping proteins (e.g., GAPDH, β-actin) to normalize ANXA11 expression.

• Molecular weight markers: Include protein ladders to confirm the detected band is of the expected size for ANXA11 (approximately 56 kDa).

• Reproducibility controls: Perform technical replicates to ensure consistent results across multiple experiments.

What methodologies are optimal for confirming ANXA11 antibody identity and specificity?

For researchers requiring rigorous confirmation of ANXA11 antibody identity and specificity, several advanced methodological approaches can be employed:

MALDI-TOF MS Fingerprinting:
This technique provides unambiguous antibody identification without lengthy sample preparation. The process involves:

• Antibody enrichment and clean-up (approximately 10 μg per sample)

• Formic acid hydrolysis to generate peptide fragments

• Generation of peptide mass fingerprints using MALDI-TOF MS

• Comparison of fingerprint patterns to identify antibody-specific peak signatures

This method is especially valuable when obtaining antibodies from third parties, as it allows researchers to "undoubtedly identify an antibody to guarantee the traceability of any research activity" . The fingerprinting approach can detect unique peptide signatures (highlighted by the five most intense signals that only occur in the fingerprint spectrum of the respective antibody), enabling reliable antibody identification without requiring full peptide sequence assignment .

Software-Assisted Analysis:
Specialized software tools like "ABID" can assist in managing antibody fingerprint data:

• Create libraries of antibody fingerprint spectra

• Automatically identify peaks and generate mass lists

• Compare new antibody samples against library entries

• Calculate match probabilities based on peak correlation

Epitope Mapping:
For ANXA11 antibodies, determining the specific binding epitope can provide crucial information:

• Use peptide arrays covering the ANXA11 sequence (Met1~Asp505)

• Apply the antibody and detect binding to specific peptide fragments

• Confirm results with mutational analysis of key residues

• Compare epitope location with known functional domains of ANXA11

How can researchers troubleshoot inconsistent results when using ANXA11 antibodies?

When encountering inconsistent results with ANXA11 antibodies, researchers should systematically address potential issues through the following troubleshooting approach:

Antibody Validation Issues:

• Confirm antibody identity using MALDI-TOF MS fingerprinting

• Verify target specificity through appropriate controls

• Assess lot-to-lot variability by comparing antibody performance across batches

• Determine optimal antibody concentration through titration experiments

Technical Factors:

• Sample preparation: Ensure complete protein extraction and prevent protein degradation with appropriate protease inhibitors

• Antigen accessibility: Optimize fixation and antigen retrieval methods for IHC/ICC applications

• Signal detection: Adjust exposure times and detection methods to capture the appropriate signal range

• Blocking conditions: Test different blocking reagents to reduce background without compromising specific signal

Experimental Design Considerations:

• Signal quantification: Implement appropriate normalization strategies for quantitative comparisons

• Reproducibility: Perform technical and biological replicates to assess experimental variability

• Positive and negative controls: Include appropriate controls in each experiment

• Cross-validation: Confirm results using alternative methods (e.g., mRNA expression, mass spectrometry)

Sample-Specific Issues:

• Post-translational modifications: Consider that modifications of ANXA11 might affect antibody recognition

• Protein isoforms: Verify which ANXA11 isoforms are recognized by the antibody

• Species differences: Ensure the antibody is validated for the species being studied

• Tissue/cell-specific factors: Account for matrix effects or endogenous blocking factors

What are the current experimental approaches for studying ANXA11's role in disease pathogenesis?

Current research on ANXA11's role in disease pathogenesis employs several sophisticated experimental approaches:

Genetic Manipulation Studies:

• CRISPR/Cas9-mediated knockout or knockin of ANXA11 in cell lines

• RNAi-mediated knockdown to study partial loss of function

• Overexpression of wild-type or mutant ANXA11 to assess gain-of-function effects

• Generation of transgenic animal models with altered ANXA11 expression

Protein-Protein Interaction Analysis:

• Co-immunoprecipitation using ANXA11 antibodies to identify binding partners

• Proximity labeling techniques (BioID, APEX) to map the ANXA11 interactome

• Yeast two-hybrid screening to identify novel interactions

• Fluorescence resonance energy transfer (FRET) to study dynamic interactions in living cells

Functional Assays:

• Calcium-dependent membrane binding assays to assess ANXA11 translocation

• Vesicle trafficking analysis using live-cell imaging

• Cell cycle progression and apoptosis assays to evaluate ANXA11's role in these processes

• Stress response assays to determine ANXA11's function under various cellular stresses

Clinical Correlation Studies:

• Immunohistochemical analysis of ANXA11 expression in patient samples

• Correlation of ANXA11 levels with disease severity or progression

• Analysis of ANXA11 variants in patient cohorts

• Evaluation of ANXA11 as a potential biomarker for disease diagnosis or prognosis

How can mass spectrometry techniques be integrated with antibody-based detection of ANXA11?

Integration of mass spectrometry with antibody-based detection creates powerful approaches for ANXA11 research:

Immunoprecipitation-Mass Spectrometry (IP-MS):

• Immunoprecipitate ANXA11 using validated antibodies

• Process samples for MS analysis through digestion with trypsin

• Perform LC-MS/MS to identify ANXA11 and its interacting partners

• Quantify relative abundances of proteins in the immunoprecipitated complex

This combined approach allows researchers to confirm ANXA11 antibody specificity while simultaneously identifying novel protein interactions and post-translational modifications.

Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM):

• Develop targeted MS assays for specific ANXA11 peptides

• Use antibody-based enrichment to increase ANXA11 concentration prior to MS analysis

• Quantify ANXA11 abundance with high specificity and sensitivity

• Detect specific post-translational modifications on ANXA11

Antibody Validation by MS:
The MALDI-TOF MS fingerprinting methodology described in the literature provides a rapid approach for antibody validation:

• Clean-up and enrichment of IgG antibodies

• Measurement of antibody concentration using Bradford Assay

• Formic acid hydrolysis (generating distinctive peptide patterns)

• MALDI-TOF MS analysis to create peptide mass fingerprints

• Comparison with reference spectra for identification

This method can be used to confirm the identity of ANXA11 antibodies obtained from third parties or to track antibody consistency across experiments .

What are the recommended protocols for quantitative analysis of ANXA11 expression using antibody-based techniques?

For reliable quantitative analysis of ANXA11 expression, researchers should implement the following protocols:

Quantitative Western Blotting:

• Sample preparation: Standard lysis with protease inhibitors

• Protein quantification: Bradford Assay to ensure equal loading

• Standard curve generation: Include a dilution series of recombinant ANXA11

• Loading controls: Include housekeeping proteins for normalization

• Signal detection: Use digital imaging systems with appropriate dynamic range

• Densitometric analysis: Measure band intensity using specialized software

• Normalization: Calculate relative ANXA11 expression normalized to loading controls

• Statistical analysis: Perform appropriate statistical tests for comparing groups

Quantitative Immunohistochemistry:

• Tissue processing: Standardize fixation and antigen retrieval

• Staining protocol: Use automated staining platforms for consistency

• Controls: Include positive and negative controls on each slide

• Digital image acquisition: Capture images under consistent conditions

• Computer-assisted analysis: Use specialized software for quantifying staining intensity

• Region of interest selection: Define consistent regions for analysis

• Scoring system development: Implement H-score or Allred score for semi-quantitative analysis

• Blind assessment: Have multiple observers score slides independently

Flow Cytometry:

• Cell preparation: Optimize fixation and permeabilization for intracellular ANXA11 staining

• Antibody titration: Determine optimal concentration for maximal signal-to-noise ratio

• Multi-parameter analysis: Include markers to identify relevant cell populations

• Controls: Incorporate isotype controls and unstained samples

• Compensation: Adjust for spectral overlap when using multiple fluorophores

• Data acquisition: Collect sufficient events for statistical analysis

• Gating strategy: Implement consistent gating approach across samples

• Quantification: Report median fluorescence intensity or percent positive cells

ELISA for ANXA11:

• Plate coating: Use capture antibody at optimized concentration

• Blocking: Reduce non-specific binding with appropriate blocking buffer

• Standard curve: Include recombinant ANXA11 standards

• Sample dilution: Test multiple dilutions to ensure measurements within linear range

• Detection: Use conjugated detection antibody or secondary antibody system

• Signal development: Optimize timing for consistent results

• Data analysis: Generate standard curve using appropriate regression method

• Quality control: Include internal controls to assess plate-to-plate variability

How is ANXA11 antibody research contributing to understanding neurodegenerative diseases?

ANXA11 antibody-based research has revealed important connections between ANXA11 and neurodegenerative diseases. Immunohistochemical studies using ANXA11 antibodies have identified altered expression patterns in neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These studies have demonstrated that ANXA11 participates in RNA granule dynamics and stress granule formation, processes critical to neuronal function and survival. Through immunoprecipitation experiments, researchers have identified ANXA11 interactions with proteins involved in RNA metabolism and vesicle trafficking, providing insight into disease mechanisms. Additionally, analysis of patient samples using ANXA11 antibodies has revealed that ANXA11 mutations can lead to protein aggregation and disruption of calcium homeostasis, contributing to neurodegeneration. ANXA11 antibody studies have also shown that the protein plays roles in autophagy and calcium-dependent vesicular transport, functions that may be compromised in neurodegenerative diseases.

What are the emerging techniques for detecting post-translational modifications of ANXA11?

Detection of post-translational modifications (PTMs) on ANXA11 is critical for understanding its functional regulation. Several emerging techniques combine antibody-based approaches with advanced analytical methods:

• Phospho-specific antibodies: Development of antibodies recognizing specific phosphorylated residues on ANXA11 enables direct detection of this important regulatory modification.

• Combined IP-MS approach: Immunoprecipitation with ANXA11 antibodies followed by mass spectrometry analysis allows comprehensive mapping of multiple PTMs simultaneously.

• Proximity ligation assay (PLA): This technique can detect specific ANXA11 modifications in situ with high sensitivity by using antibodies against both ANXA11 and the modification of interest.

• Phos-tag SDS-PAGE: When combined with ANXA11 antibodies for Western blotting, this specialized electrophoresis technique improves separation of phosphorylated ANXA11 forms.

• ELISA-based PTM detection: Development of sandwich ELISA systems using ANXA11 capture antibodies and PTM-specific detection antibodies enables quantitative assessment of modified ANXA11.

• MALDI-imaging mass spectrometry: This technique allows spatial visualization of ANXA11 PTMs in tissue sections when combined with antibody-based enrichment strategies.

How can ANXA11 antibodies be employed in high-throughput screening applications?

ANXA11 antibodies can be adapted for various high-throughput screening approaches:

Automated Immunocytochemistry:

• Culture cells in 96- or 384-well plates

• Perform automated fixation, permeabilization, and staining with ANXA11 antibodies

• Use high-content imaging systems for automated image acquisition

• Implement image analysis algorithms to quantify ANXA11 expression, localization, or aggregation

• Screen compounds for effects on ANXA11 biology

Reverse Phase Protein Array (RPPA):

• Spot cell or tissue lysates onto nitrocellulose-coated slides

• Probe with validated ANXA11 antibodies

• Use signal amplification systems for detection

• Analyze large sample sets simultaneously for ANXA11 expression

• Correlate findings with clinical outcomes or experimental conditions

Multiplexed Bead-Based Assays:

• Couple ANXA11 antibodies to uniquely coded microbeads

• Incubate with samples and detection antibodies

• Analyze using flow cytometry-based platforms

• Simultaneously measure ANXA11 alongside other proteins of interest

• Process hundreds of samples with minimal sample consumption

These high-throughput approaches enable efficient screening of drug candidates, genetic modifiers, or disease biomarkers related to ANXA11 function, significantly accelerating discovery in this field.