ANXA4 Recombinant Monoclonal Antibody

What is Annexin-4/ANXA4 and what are its primary cellular functions?

Annexin-4 (ANXA4) is a calcium/phospholipid-binding protein that primarily promotes membrane fusion and plays significant roles in exocytosis processes . It belongs to the annexin family of proteins, which are characterized by their ability to bind phospholipids in a calcium-dependent manner. ANXA4 has several alternative names in the literature, including 35-beta calcimedin, Annexin IV, carbohydrate-binding protein p33/p41, chromobindin-4, endonexin I, lipocortin IV, placental anticoagulant protein II, and protein II (PAP-II) . The protein associates with the inner surface of the plasma membrane and participates in various cellular processes including vesicle trafficking and membrane dynamics . In research contexts, ANXA4 is frequently studied for its potential roles in cellular signaling pathways and has been implicated in certain disease processes, making it an important target for antibody-based detection methods.

What laboratory applications are ANXA4 recombinant monoclonal antibodies suitable for?

ANXA4 recombinant monoclonal antibodies demonstrate versatility across multiple laboratory applications. Based on validated research data, these antibodies are suitable for Western blotting (WB), immunohistochemistry on paraffin-embedded tissues (IHC-P), immunocytochemistry/immunofluorescence (ICC/IF), immunohistochemistry on frozen sections (IHC-Fr), and intracellular flow cytometry (Flow Cyt Intra) . Some ANXA4 antibodies, such as polyclonal variants, are also applicable for immunoprecipitation (IP) . When selecting an ANXA4 antibody for specific applications, researchers should consider whether the antibody has been validated for their particular application. For example, the rabbit recombinant monoclonal ANXA4 antibody (EPR22929-208) has been extensively validated across multiple applications and species, providing researchers with high confidence in experimental outcomes across diverse methodological approaches .

What species reactivity can be expected from commercially available ANXA4 antibodies?

Species reactivity is a critical consideration when selecting antibodies for research. Available ANXA4 antibodies demonstrate varying cross-reactivity profiles depending on their development and validation. Polyclonal ANXA4 antibodies like ab153883 have been primarily validated for reactivity with human samples . In contrast, recombinant monoclonal antibodies such as EPR22929-208 (ab256456) offer broader species reactivity, having been validated for use with human, mouse, and rat samples . This extended cross-reactivity makes recombinant monoclonal antibodies particularly valuable for comparative studies across different model organisms. The cross-reactivity is supported by experimental validation, including Western blot analyses showing positive detection in human tissues (HepG2, HeLa), mouse tissues (liver, kidney, lung), and rat tissues (kidney) . When planning experiments involving multiple species, researchers should carefully review validation data to ensure proper antibody selection.

How should researchers determine the optimal antibody dilution for different applications?

Determining the optimal antibody dilution is essential for achieving specific signal while minimizing background. The process should begin with manufacturer recommendations as baseline starting points, followed by empirical optimization for each specific application and experimental system. For the rabbit recombinant monoclonal ANXA4 antibody, validated dilutions include 1/5000 for IHC-P (equivalent to 0.103 μg/ml), 1/100 for IHC-Fr, and 1/500 for flow cytometry . For Western blotting applications, a 1/1000 dilution has been found effective for polyclonal antibodies . Optimization typically involves testing a range of dilutions (e.g., 1/250, 1/500, 1/1000, 1/2000) and selecting the concentration that provides the best signal-to-noise ratio. Factors affecting optimal dilution include tissue type, fixation method, antigen abundance, and detection system sensitivity. Researchers should document optimization experiments thoroughly, including positive and negative controls, to ensure reproducibility across experimental batches.

What factors influence the specificity and sensitivity of ANXA4 recombinant monoclonal antibodies in different experimental contexts?

Multiple factors impact the specificity and sensitivity of ANXA4 recombinant monoclonal antibodies across experimental contexts. First, epitope accessibility varies significantly depending on sample preparation methods. For instance, in immunohistochemistry applications, optimal epitope exposure requires specific antigen retrieval methods, commonly heat-mediated retrieval with citrate buffer (pH 6.0) . Second, fixation protocols dramatically affect epitope preservation and accessibility—paraformaldehyde fixation (4%) combined with permeabilization using Triton X-100 (0.1-0.2%) has been validated for immunofluorescence applications with ANXA4 antibodies . Third, blocking conditions significantly impact background signal; insufficient blocking can lead to nonspecific binding while excessive blocking might mask specific signals. Fourth, detection system sensitivity influences the antibody's practical detection limit, with amplification systems (like tyramide signal amplification) potentially enhancing sensitivity for low-abundance targets. Finally, tissue-specific factors can affect antibody performance; ANXA4 expression patterns differ significantly across tissues, with documented expression in human endometrium, kidney, pancreas, and other tissues . Understanding these variables allows researchers to optimize protocols for specific experimental questions and sample types.

What are the optimal antigen retrieval methods for ANXA4 immunohistochemistry?

For ANXA4 immunohistochemistry, heat-mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 minutes has been experimentally validated as optimal . This method effectively unmasks ANXA4 epitopes in paraffin-embedded tissues while preserving tissue morphology. The retrieval process disrupts protein cross-links formed during fixation, allowing antibodies to access the target epitopes. For researchers implementing this protocol, several technical considerations warrant attention: first, consistent temperature maintenance during the retrieval process is critical for reproducible results; second, complete coverage of tissue sections with retrieval solution prevents drying artifacts; third, allowing appropriate cool-down periods after heat treatment prevents tissue detachment from slides. For frozen tissue sections, milder retrieval methods are typically sufficient, such as brief incubation with sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20) . Importantly, regardless of the retrieval method, researchers should validate the protocol with positive and negative controls for their specific tissue type and fixation conditions, as optimal retrieval conditions can vary based on tissue-specific factors and fixation duration.

What controls should be included when using ANXA4 antibodies for various applications?

Rigorous experimental design for ANXA4 antibody applications requires comprehensive controls to validate results and facilitate accurate interpretation. Primary antibody controls should include: (1) Positive tissue/cell controls known to express ANXA4, such as HepG2 cells for Western blotting or human endometrium for IHC ; (2) Negative tissue/cell controls where ANXA4 expression is minimal or absent; (3) Isotype controls using non-specific antibodies of the same isotype and concentration as the ANXA4 antibody (e.g., Rabbit monoclonal IgG isotype control) ; and (4) Antibody titration controls to demonstrate dose-dependent signal intensity. Secondary antibody controls should include: (1) Secondary-only controls omitting primary antibody to assess non-specific binding of the secondary antibody, as exemplified in the validated protocols using Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ; and (2) Autofluorescence controls for fluorescence-based detection methods. Additional validation controls might include peptide competition assays, where pre-incubation of the antibody with purified ANXA4 protein should abolish specific staining, and knockout/knockdown controls in appropriate model systems. Implementing this comprehensive control strategy ensures that observed signals genuinely represent ANXA4 expression rather than technical artifacts.

What are the most effective sample preparation methods for detecting ANXA4 using Western blotting?

Effective Western blotting for ANXA4 detection requires optimized sample preparation protocols to ensure protein integrity and epitope accessibility. Cell or tissue lysis should be performed using buffers containing protease inhibitors to prevent ANXA4 degradation. For Western blotting applications, validated protocols have utilized whole cell lysates from various sources including HepG2 cells (human hepatocellular carcinoma), HeLa cells (human cervix adenocarcinoma), and tissue lysates from liver, kidney, and lung of multiple species . Sample loading typically ranges from 20-30 μg of total protein per lane, with separation on 10% SDS-PAGE gels . ANXA4 detection reveals characteristic bands at approximately 31-37 kDa, with reported observed band sizes of 31 kDa and 36 kDa or predicted band sizes of 35 kDa and 37 kDa . Complete protein transfer to membranes is critical and can be verified using reversible total protein stains. Blocking should be optimized to minimize background while preserving specific signal, typically using 5% non-fat dry milk or bovine serum albumin. For detection, enhanced chemiluminescence systems provide suitable sensitivity, with antibody dilutions ranging from 1/1000 to 1/100000 depending on the specific antibody and detection system .

How should researchers interpret multiple band patterns when using ANXA4 antibodies in Western blotting?

Multiple band patterns in ANXA4 Western blotting require systematic interpretation to distinguish genuine isoforms from artifacts. ANXA4 antibodies typically detect bands around 31-37 kDa, with specifically observed patterns including 31 kDa and 36 kDa bands with recombinant monoclonal antibodies and predicted band sizes of 35 kDa and 37 kDa with polyclonal antibodies . When multiple bands appear, researchers should first consider whether they represent known ANXA4 isoforms or post-translational modifications. The presence of multiple bands within the expected size range may indicate physiologically relevant protein variants. Second, researchers should systematically rule out technical artifacts by examining lysate preparation methods (inadequate denaturation can cause aggregation or incomplete resolution), checking for proteolytic degradation (adding fresh protease inhibitors can prevent this), and validating specificity through knockdown/knockout controls. Third, tissue-specific expression patterns should be considered, as ANXA4 expression varies across tissues and may present different isoform distributions. Quantitative analysis of multiple bands should be approached cautiously, with normalization to appropriate loading controls and consideration of total ANXA4 signal across all specific bands when comparing expression levels between samples.

What strategies can address contradictory results when using different ANXA4 antibody clones?

When faced with contradictory results from different ANXA4 antibody clones, researchers should implement a systematic troubleshooting approach to resolve discrepancies. First, epitope mapping should be conducted to determine whether different antibodies recognize distinct regions of the ANXA4 protein, which might explain differential detection patterns if certain epitopes are masked in specific contexts. Second, validation using orthogonal methods is essential—complementary techniques like mass spectrometry, RNA expression analysis (RT-PCR or RNA-seq), or genetic models (knockdown/knockout) can provide antibody-independent confirmation of ANXA4 expression patterns. Third, comprehensive specificity testing should be performed, including peptide competition assays and immunoprecipitation followed by mass spectrometry to confirm target identity. Fourth, tissue-specific factors should be considered, as certain cellular environments might affect epitope accessibility differently for various antibody clones. Fifth, technical optimization for each antibody clone should be conducted independently, as optimal conditions often differ between clones . When reporting contradictory results, researchers should transparently document all antibodies used (including clone identifiers and catalog numbers), precisely describe the experimental conditions for each, and discuss possible explanations for observed differences. This approach not only resolves immediate experimental questions but also contributes valuable information to the broader research community.

What emerging technologies are enhancing the specificity and utility of ANXA4 recombinant monoclonal antibodies?

Recombinant antibody technology represents a significant advancement in ANXA4 research, offering unprecedented consistency and specificity compared to traditional monoclonal and polyclonal approaches . These engineered antibodies are produced using recombinant DNA technology, ensuring defined genetic composition and eliminating the batch-to-batch variability inherent in traditional antibody production methods. Current technological advances continue to enhance ANXA4 antibody utility through several approaches: First, epitope-focused engineering enables the creation of antibodies targeting specific ANXA4 regions with functional significance, allowing more precise investigation of structure-function relationships. Second, multiparameter analysis capabilities are expanding through conjugation with diverse fluorophores and nanoparticles, facilitating complex flow cytometry and imaging applications. Third, recombinant antibody fragments (Fab, scFv) offer advantages in certain applications through improved tissue penetration and reduced background. Fourth, humanized ANXA4 antibodies are being developed for potential therapeutic applications based on research findings. Future directions in this field include the development of antibodies specifically targeting post-translational modifications of ANXA4, conditional detection systems responsive to ANXA4 activation states, and integration with emerging single-cell analysis technologies for investigating ANXA4 expression at unprecedented resolution.