AMOT Antibody

What is AMOT and why is it important in research?

Angiomotin (AMOT) belongs to the motin family of angiostatin-binding proteins, characterized by conserved coiled-coil domains and C-terminal PDZ binding motifs. The protein is predominantly expressed in endothelial cells of capillaries and larger vessels, particularly in the placenta, where it mediates inhibitory functions. AMOT serves as a receptor for angiostatin and plays crucial roles in regulating cell polarity, migration, and angiogenesis processes. The significance of AMOT in research stems from its involvement as a key regulator of cell signaling pathways that control growth and survival in response to changes in the cellular microenvironment. Dysregulation of AMOT has been linked to tumorigenesis and progression in various cancers, making it a promising target for therapeutic interventions in oncology research. Understanding AMOT's biological functions provides insights into fundamental cellular processes related to cancer, cardiovascular disease, and developmental biology.

What are the validated applications for AMOT antibodies?

AMOT antibodies have been validated for multiple experimental applications, providing researchers with versatile tools for investigating this protein across different platforms. Western blot (WB) analysis represents one of the primary applications, with recommended dilutions ranging from 1:500-1:9000, depending on the specific antibody product and sample type. Immunoprecipitation (IP) has been validated using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate, particularly effective with HEK-293 cells. For immunohistochemistry (IHC), AMOT antibodies perform optimally at dilutions between 1:50-1:500, with positive detection demonstrated in human colon cancer tissue. Immunofluorescence (IF) and immunocytochemistry (ICC) applications have been validated at 1:50-1:500 dilutions, with successful detection in HEK-293 cells. Additionally, some AMOT antibodies have been validated for ELISA applications, enabling quantitative assessment of AMOT protein levels in research samples.

What sample types have been validated for AMOT antibody detection?

Several sample types have been validated for reliable AMOT detection using specific antibodies. For Western blot applications, human cell lines including HEK-293 and HEK-293T cells have demonstrated positive detection of AMOT. In mouse models, lung tissue serves as a reliable positive sample for AMOT detection, as indicated in validation studies. For immunohistochemistry applications, human colon cancer tissue has been validated as a positive control, requiring specific antigen retrieval conditions (preferably with TE buffer pH 9.0, or alternatively with citrate buffer pH 6.0). For immunoprecipitation experiments, HEK-293 cells provide a reliable source of AMOT protein that can be successfully immunoprecipitated with anti-AMOT antibodies. When establishing new protocols or working with untested sample types, researchers should first validate antibody performance using these confirmed positive samples as controls. Optimization of protocols may be necessary when expanding to different tissue types or experimental models not previously validated.

How does AMOT antibody validation differ from other antibody validations?

AMOT antibody validation requires special considerations due to its multiple isoforms and varied expression patterns across tissues. Unlike antibodies targeting more uniformly expressed proteins, AMOT antibody validation should include isoform-specific controls to confirm detection of the correct 80 kDa and/or 130 kDa variants. Researchers should implement a multi-parameter validation approach that includes positive and negative controls through gene overexpression and knockdown/knockout systems respectively. For example, transfection of HEK-293 cells with AMOT expression vectors serves as an excellent positive control, while CRISPR/Cas9-mediated AMOT knockout cells provide definitive negative controls. Cross-reactivity testing is particularly important since AMOT belongs to a family of related proteins including AMOTL1 and AMOTL2 with significant homology. Application-specific validation is essential—while an antibody may perform well in Western blot, its performance in immunohistochemistry or immunofluorescence cannot be presumed without explicit testing. Batch-to-batch consistency assessments using standardized positive samples (such as mouse lung tissue or HEK-293 cells) provide quality assurance for long-term experimental reproducibility.

How can researchers use AMOT antibodies to study tumor angiogenesis?

AMOT antibodies serve as powerful tools for investigating tumor angiogenesis through multiple experimental approaches. Researchers can utilize immunohistochemistry with AMOT antibodies at 1:50-1:500 dilutions to visualize AMOT expression patterns in tumor vasculature compared to normal tissue vasculature, enabling quantification of AMOT upregulation during tumorigenesis. Dual immunofluorescence staining combining AMOT antibodies with endothelial markers (CD31/PECAM-1) allows precise localization of AMOT expression within the tumor vasculature microenvironment. Western blot analysis comparing AMOT expression levels between normal endothelial cells and tumor-associated endothelial cells provides quantitative assessment of expression changes during malignant progression. In functional studies, blocking AMOT with neutralizing antibodies in endothelial cell migration and tube formation assays directly assesses its role in angiogenic processes. Co-immunoprecipitation experiments using AMOT antibodies can identify binding partners specific to tumor endothelial cells, revealing context-specific signaling mechanisms. These approaches collectively enable researchers to determine how AMOT contributes to tumor angiogenesis, vessel permeability alterations, and potential therapeutic vulnerabilities.

What methodologies can detect AMOT-mediated effects on vessel permeability?

Detecting AMOT-mediated effects on vessel permeability requires specialized methodologies that measure vascular integrity and barrier function. Researchers can employ transwell permeability assays with endothelial cell monolayers treated with anti-AMOT antibodies to directly measure macromolecule passage across the endothelial barrier in vitro. In vivo assays using Evans Blue dye or fluorescent-labeled dextrans following anti-AMOT administration quantitatively assess vascular leakage in tumor models. Immunohistochemical analysis of tumor sections after anti-AMOT treatment allows visualization of morphological changes in vessel structure, including increased vessel diameter and formation of lacunar spaces, as documented in studies where anti-AMOT antibodies led to "massive tumor perivascular necrosis" associated with altered vessel permeability. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) provides non-invasive monitoring of tumor vessel permeability changes following AMOT targeting in preclinical models. Researchers should incorporate permeability measurements when evaluating combination therapies, as studies have shown that "greater tumor vessel permeability also markedly enhances the antitumor effect of doxorubicin" following AMOT targeting, suggesting important implications for improving chemotherapy delivery through modulation of vessel permeability.

How do anti-AMOT antibodies affect tumor vasculature differently from other anti-angiogenic approaches?

Anti-AMOT antibodies demonstrate distinct mechanisms of action compared to traditional anti-angiogenic therapies such as VEGF inhibitors. While VEGF-targeted therapies primarily prevent new vessel formation, anti-AMOT antibodies have been shown to alter the structure and function of existing tumor vasculature, causing increased vessel diameter with formation of lacunar spaces and enhanced permeability. This morphological remodeling leads to distinctive perivascular necrosis patterns not typically observed with VEGF inhibition. Anti-AMOT approaches appear to preserve normal vessel function while selectively targeting tumor vasculature, potentially offering reduced systemic toxicity compared to pan-angiogenesis inhibitors. Evidence indicates that anti-AMOT antibodies trigger immune-mediated mechanisms including "an effective epitope spreading that induces an immune response against other tumor associated antigens," which represents an immunological dimension absent in traditional anti-angiogenic approaches. The enhanced permeability effect of anti-AMOT treatment creates a synergistic opportunity with chemotherapy, as studies show it "markedly enhances the antitumor effect of doxorubicin" by improving drug delivery to tumor tissues. These multifaceted effects position anti-AMOT strategies as potentially complementary rather than redundant to existing anti-angiogenic therapeutic approaches.

What are the optimal conditions for Western blot detection of AMOT?

Achieving optimal Western blot detection of AMOT requires careful consideration of sample preparation, electrophoresis conditions, and detection parameters. Researchers should prepare protein lysates using RIPA buffer supplemented with protease inhibitors, phosphatase inhibitors, and deubiquitinase inhibitors to preserve AMOT's post-translational modifications. Protein denaturation should be performed at 70°C for 10 minutes rather than boiling, as high temperatures may cause AMOT aggregation that impairs migration. Separation should be conducted on 7-8% polyacrylamide gels to achieve optimal resolution of both the 80 kDa and 130 kDa AMOT isoforms, with extended run times to properly resolve these high-molecular-weight proteins. After transfer to PVDF membranes (which perform better than nitrocellulose for AMOT detection), blocking should be performed with 5% non-fat dry milk in TBST for 2 hours at room temperature. For primary antibody incubation, AMOT antibodies should be diluted in the range of 1:1000-1:9000 in 5% BSA/TBST and incubated overnight at 4°C with gentle agitation to ensure uniform binding. Following stringent washing steps (at least 3 x 10 minutes with TBST), HRP-conjugated secondary antibodies should be applied at 1:5000-1:10000 dilutions for 1 hour at room temperature. Enhanced chemiluminescence detection systems with extended exposure times may be necessary for visualizing lower-abundance isoforms.

What protocols are recommended for immunohistochemical detection of AMOT?

For successful immunohistochemical detection of AMOT in tissue samples, researchers should follow a specialized protocol that accounts for AMOT's susceptibility to fixation artifacts and epitope masking. Tissue samples should be fixed in 10% neutral-buffered formalin for no more than 24 hours to prevent overfixation, followed by paraffin embedding using standard procedures. Sections should be cut at 4-5 μm thickness and mounted on positively charged slides. Heat-induced epitope retrieval is critical, with optimal results achieved using TE buffer at pH 9.0 (while citrate buffer at pH 6.0 serves as an alternative but less effective option) in a pressure cooker for 20 minutes. Following cooling and PBS washing, endogenous peroxidase blocking should be performed using 3% hydrogen peroxide for 10 minutes. Non-specific binding should be minimized with a 5% normal goat serum block for 1 hour at room temperature. AMOT antibody application should be performed at dilutions between 1:50-1:500 (with 1:200 often providing optimal signal-to-noise ratio) overnight at 4°C in a humidified chamber. Detection systems utilizing polymer-based secondary antibodies provide superior sensitivity compared to standard ABC methods. DAB (3,3'-diaminobenzidine) should be applied for 5-7 minutes with careful monitoring to prevent overdevelopment, followed by hematoxylin counterstaining for 30 seconds. Positive controls (human colon cancer tissue) and negative controls (primary antibody omission) should be included in each staining run to validate specificity.

How can researchers quantify AMOT antibody responses in immunization experiments?

Quantifying AMOT antibody responses in immunization experiments requires rigorous serological analysis techniques that ensure accurate measurement of both antibody titers and functional activity. Researchers should implement ELISA assays using recombinant AMOT protein (or specific AMOT peptides) as the coating antigen at 1-2 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C. After blocking with 5% BSA in TBS-Tween buffer for 2 hours, serially diluted serum samples should be applied (starting at 1:100 and performing 2-fold dilutions) and incubated for 2 hours at room temperature. Detection can be performed using species-appropriate HRP-conjugated secondary antibodies (typically anti-mouse or anti-rat IgG) at 1:5000 dilution for 1 hour, followed by TMB substrate development and optical density measurement at 450 nm. For more comprehensive analysis, isotype-specific quantification should be performed using biotin-conjugated anti-mouse IgG1, IgG2a, IgG2b, and IgG3 secondary antibodies (1:1000 dilution) followed by streptavidin-HRP (1:200 dilution) to characterize the nature of the immune response (Th1 vs. Th2 bias). Functional analysis of anti-AMOT antibodies should include in vitro assessment of their ability to inhibit endothelial cell migration and tube formation, as well as their capacity to block tumor cell- and basic fibroblast growth factor-induced angiogenesis in matrigel plug assays. These multifaceted analyses collectively provide a comprehensive profile of anti-AMOT immune responses.

What criteria should guide selection of an appropriate AMOT antibody for specific applications?

Selecting the appropriate AMOT antibody requires systematic evaluation of multiple technical parameters to ensure optimal performance in specific applications. Researchers should first consider antibody specificity—polyclonal antibodies offer broader epitope recognition but potentially higher background, while monoclonal antibodies provide consistency but might miss certain isoforms or conformational states of AMOT. Epitope location is crucial; antibodies targeting the N-terminal region detect primarily the p130 isoform, while C-terminal-targeting antibodies detect both p80 and p130 isoforms. Cross-reactivity with related proteins (AMOTL1 and AMOTL2) should be explicitly evaluated, especially when studying tissues where multiple motin family members are expressed. Application-specific validation data should be thoroughly reviewed—antibodies performing well in Western blot may not necessarily work in immunohistochemistry or immunoprecipitation. Species reactivity must align with experimental models; some antibodies react only with human AMOT while others cross-react with mouse and rat orthologs. Technical aspects including recommended dilutions, storage requirements, and formulation (BSA-containing formulations may be problematic for certain applications) should be evaluated within the context of planned experiments. Publication history provides valuable evidence of real-world performance, with antibodies referenced in peer-reviewed studies offering greater confidence in their reliability and reproducibility across different experimental settings.

How should researchers validate AMOT antibody specificity?

Comprehensive validation of AMOT antibody specificity requires a multi-pronged approach that systematically eliminates potential false positive and false negative scenarios. Researchers should begin with Western blot analysis using positive control samples (such as HEK-293 cells or mouse lung tissue) to confirm detection of the appropriate 80 kDa and 130 kDa bands. Genetic validation through siRNA/shRNA knockdown or CRISPR/Cas9 knockout models provides definitive evidence of specificity when the anticipated bands disappear following AMOT depletion. Conversely, overexpression systems using transfected AMOT expression vectors should demonstrate increased signal intensity at the expected molecular weights. Peptide competition assays, where the antibody is pre-incubated with the immunizing peptide before application to the sample, should abolish specific signals if the antibody is truly targeting AMOT. Cross-reactivity with related proteins (AMOTL1/AMOTL2) should be examined using recombinant protein standards or cells expressing only specific family members. For immunohistochemistry/immunofluorescence applications, co-localization studies with known AMOT binding partners or subcellular markers should demonstrate expected patterns (e.g., tight junction localization). Parallel testing with multiple AMOT antibodies recognizing different epitopes provides additional confirmation when they yield consistent results despite their different binding regions. These systematic validation steps collectively establish a robust foundation for confident interpretation of experimental results.

How does AMOT expression correlate with tumor progression and clinical outcomes?

AMOT expression demonstrates complex relationships with tumor progression and clinical outcomes that vary by cancer type, highlighting its context-dependent roles in malignancy. Research using AMOT antibodies has revealed increased AMOT expression on tumor endothelia during the progression from pre-neoplastic lesions to full-fledged carcinoma, particularly in breast cancer models. This progressive upregulation corresponds to increased angiogenic activity and may serve as a biomarker for malignant transformation. AMOT expression patterns often correlate with vessel abnormalities characteristic of tumor vasculature, including increased diameter, irregular morphology, and enhanced permeability which collectively contribute to altered tumor perfusion and drug delivery dynamics. The functional significance of AMOT in tumor progression has been demonstrated through anti-AMOT vaccination approaches, where the intensity of tumor inhibition "directly correlated with the titer of anti-Amot antibodies induced by the vaccine," establishing a mechanistic link between AMOT neutralization and tumor growth suppression. In therapeutic contexts, AMOT targeting appears particularly effective against autochthonous tumors in genetically engineered mouse models (BALB-neuT and PyMT) as well as transplantable tumors, suggesting broad applicability across different tumor development paradigms. These findings collectively position AMOT as both a biomarker of tumor progression and a therapeutic target with significant potential for clinical translation in oncology.

What methodologies can assess the impact of anti-AMOT approaches on tumor chemosensitivity?

Assessing the impact of anti-AMOT approaches on tumor chemosensitivity requires specialized methodologies that evaluate both vascular changes and enhanced drug delivery. Researchers should employ combination therapy protocols in preclinical models where anti-AMOT treatments (antibodies or vaccines) are administered prior to standard chemotherapeutic agents, with appropriate timing to allow for vascular remodeling before chemotherapy administration. Quantitative biodistribution studies using fluorescently-labeled or radiolabeled chemotherapeutic agents can directly measure enhanced drug accumulation in tumors following anti-AMOT treatment compared to control conditions. Intravital microscopy using window chamber models provides real-time visualization of vascular changes and drug distribution patterns within the tumor microenvironment after anti-AMOT intervention. Histopathological analysis should examine markers of chemotherapy-induced damage (γH2AX for DNA damage or cleaved caspase-3 for apoptosis) to determine whether anti-AMOT pretreatment enhances the cytotoxic effects of subsequently administered chemotherapeutics. Functional studies evaluating tumor growth kinetics and survival outcomes in response to combination therapy versus either agent alone are essential for demonstrating clinical relevance. These approaches collectively enable researchers to quantify the degree to which "greater tumor vessel permeability also markedly enhances the antitumor effect of doxorubicin" and potentially other chemotherapeutic agents following anti-AMOT treatment, providing a rationale for "the development of novel anticancer treatments based on anti-Amot vaccination in conjunction with chemotherapy regimens."

What are common technical challenges when working with AMOT antibodies and how can they be addressed?

Researchers frequently encounter several technical challenges when working with AMOT antibodies, each requiring specific troubleshooting approaches for resolution. Detection of weak or absent signals in Western blot applications may result from insufficient protein loading or degradation; this can be addressed by increasing protein concentration (50-100 μg recommended), adding fresh protease inhibitors, and avoiding repeated freeze-thaw cycles of samples. Non-specific bands may appear due to antibody cross-reactivity or sample overloading; researchers should optimize antibody dilution (starting with 1:1000), extend blocking time to 2 hours, and include positive control samples for band identification. Inconsistent detection of both 80 kDa and 130 kDa isoforms often occurs due to their differential expression across tissues; researchers should optimize protein extraction methods specifically for membrane proteins and utilize longer gel separation times to improve resolution. In immunohistochemistry applications, high background staining can be minimized through extended blocking (5% normal serum for 1 hour), additional washing steps (5 x 5 minutes), and careful antibody titration starting from higher dilutions (1:500). Epitope masking due to fixation represents another common challenge; researchers should prioritize TE buffer (pH 9.0) for antigen retrieval as specified in validation data, with extended retrieval times (20-30 minutes) for difficult samples. For immunoprecipitation experiments, poor pull-down efficiency can be improved by increasing antibody amount (2-4 μg per mg of lysate) and extending incubation times to overnight at 4°C with gentle rotation.

How can researchers optimize AMOT detection in challenging tissue types?

Optimizing AMOT detection in challenging tissue types requires specialized approaches that address tissue-specific barriers to antibody performance. For tissues with high endogenous biotin (such as liver, kidney, and adipose), researchers should implement avidin-biotin blocking steps prior to primary antibody application or switch to non-biotin detection systems such as polymer-based methods to prevent false positive signals. Tissues with high autofluorescence (including brain, spleen, and tissues containing lipofuscin) require additional pretreatment with Sudan Black B (0.1% in 70% ethanol for 20 minutes) or commercial autofluorescence quenchers when performing immunofluorescence detection of AMOT. For heavily fibrous tissues like breast tumors or fibrotic lesions, extended protease digestion (using proteinase K for 5-10 minutes) followed by heat-induced epitope retrieval may be necessary to improve antibody accessibility to AMOT epitopes. Highly vascularized tissues with endogenous peroxidase activity require more stringent peroxidase blocking (3% hydrogen peroxide for 15-20 minutes) to prevent non-specific DAB precipitation. When working with archival or overfixed tissues, sequential epitope retrieval combining both heat-mediated (TE buffer, pH 9.0) and enzymatic methods may retrieve masked AMOT epitopes more effectively than either method alone. Signal amplification systems such as tyramide signal amplification can enhance detection sensitivity in tissues with low AMOT expression, though careful titration is necessary to maintain specificity while improving signal strength.