AEE10 Antibody

What is AEE10 antibody and what is its primary target?

AEE10 antibody (catalog number BT1191305) is a research-grade antibody that specifically targets the probable acyl-activating enzyme 10 protein. This target protein is believed to function as an acyl-CoA synthetase, which activates carboxylic acids through acyl-CoA formation in biological systems. The antibody recognizes the protein encoded by the At1g21530 gene in Arabidopsis thaliana, also known as AMP-binding protein 10 (AMPBP10) or AtAMPBP10. When designing experiments, researchers should note that the antibody is supplied in liquid form with a buffer composition of 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% ProClin 300 as a preservative.

What are the fundamental principles of antibody specificity that apply to AEE10 antibody?

Antibody specificity, including for AEE10, relies on the precise molecular recognition between the antibody's variable regions and the target epitope. This specificity is determined by the three-dimensional structure of both molecules and their complementary binding interfaces. The principles that govern AEE10 antibody specificity are similar to those observed in other highly specific antibodies, such as phosphotyrosine antibodies, which demonstrate exquisite recognition of their targets with minimal cross-reactivity to non-phosphorylated peptides . For research applications, validation of specificity should include positive and negative controls, similar to the approach demonstrated with THE™ Phosphotyrosine Antibody (E10)plus in Western blot analyses comparing EGF-stimulated versus untreated cell lysates .

How does the UniProt annotation (Q9LPK7) help in understanding AEE10's target?

The UniProt annotation Q9LPK7 for AEE10's target provides researchers with critical information about protein structure, function, and evolutionary relationships. This database entry confirms the protein's classification as an acyl-activating enzyme and provides sequence information essential for epitope prediction. Understanding the specific regions recognized by the antibody helps researchers predict potential cross-reactivity and design blocking peptides for validation experiments. The UniProt database also connects researchers to relevant pathway data through links to complementary databases such as KEGG (ath:AT1G21530) and STRING (3702.AT1G21530.2), which provide insights into the protein's role in metabolic networks and protein-protein interactions.

What are the recommended protocols for using AEE10 antibody in Western blot applications?

For Western blot applications using AEE10 antibody, researchers should follow a methodological approach similar to that established for other specialized antibodies. Begin with sample preparation by lysing cells in a buffer containing phosphatase inhibitors (particularly important if studying post-translational modifications). Based on protocols for similar research-grade antibodies, use a protein concentration of 25-50 μg per lane . After SDS-PAGE separation and transfer to a membrane, block with 5% BSA in TBST for 1 hour at room temperature. Incubate with AEE10 antibody at a starting dilution of 1:1000, though optimization may be necessary based on signal strength . For detection, a secondary antibody system such as IRDye800 Conjugated Anti-Mouse/Rabbit IgG can be employed, similar to the detection system used with phosphotyrosine antibodies in comparative studies . Include positive control samples expressing the target protein and negative controls where the target is absent to verify specificity.

How should samples be prepared for immunocytochemistry/immunofluorescence with AEE10 antibody?

For immunocytochemistry/immunofluorescence applications with AEE10 antibody, follow these methodological guidelines: Fix cells with 4% paraformaldehyde in PBS for 15 minutes at room temperature, then permeabilize with 0.2% Triton X-100 for 10 minutes. Block non-specific binding with 1% BSA and 10% normal serum from the species of the secondary antibody for 1 hour. Based on protocols established for comparable antibodies, apply AEE10 antibody at 4 μg/ml (though this concentration should be optimized) . Incubate overnight at 4°C in a humidified chamber. For visualization, use a fluorophore-conjugated secondary antibody such as FITC-conjugated anti-mouse/rabbit IgG, incubated for 1-2 hours at room temperature protected from light . Counterstain nuclei with DAPI and mount with an anti-fade mounting medium. As with other specialized antibodies, include stimulated and unstimulated cells as positive and negative controls, respectively, to confirm antibody specificity and optimize signal-to-background ratios.

What methodological approaches can be used to optimize ELISA protocols with AEE10 antibody?

To optimize ELISA protocols with AEE10 antibody, researchers should implement a systematic methodological approach. Begin by coating high-binding ELISA plates with purified target protein or cell lysates containing the target at varying concentrations (0.1-10 μg/ml) in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C. After washing, block with 1-3% BSA in PBS for 1-2 hours at room temperature. Based on protocols for other research antibodies, test AEE10 antibody at multiple concentrations starting at 0.1 μg/ml . Following a 2-hour incubation at room temperature, use an HRP-conjugated secondary antibody and TMB substrate for detection. Develop a standard curve using purified target protein to enable quantitative analysis. To verify specificity, incorporate controls with structurally similar proteins, as demonstrated in studies with phosphotyrosine antibodies where both phosphorylated and non-phosphorylated peptides were tested to confirm absence of cross-reactivity . Optimize washing steps, antibody concentrations, and incubation times to achieve the best signal-to-noise ratio.

How can AEE10 antibody be utilized in studies investigating metabolic pathway regulation?

For investigating metabolic pathway regulation, AEE10 antibody can be employed in multi-faceted experimental approaches focusing on acyl-CoA synthetase activity. Researchers should design co-immunoprecipitation experiments to identify protein interaction partners within the fatty acid metabolism pathway, followed by mass spectrometry analysis to characterize these interactions. Combining immunofluorescence microscopy with metabolic inhibitors can reveal spatial and temporal regulation of the target protein. For comprehensive pathway analysis, researchers should implement a systems biology approach by coupling AEE10 antibody-based protein quantification with metabolomics data. This integrated approach has proven successful in studies of receptor tyrosine kinases such as EphA10, where antibody-based detection revealed not only expression patterns in tumor tissues but also interactions with immunosuppressive myeloid cells in the tumor microenvironment . Additionally, researchers can employ phosphoproteomic analysis to investigate how post-translational modifications affect AEE10 target protein activity in response to metabolic stress conditions.

What methodological considerations are important when using AEE10 antibody in comparative interspecies studies?

When conducting interspecies comparative studies with AEE10 antibody, researchers must implement a rigorous methodological framework to ensure valid cross-species comparisons. First, perform sequence alignment of the target protein across species to predict epitope conservation. Test antibody reactivity against recombinant proteins from each species before proceeding to tissue samples. When differences in reactivity are observed, quantify them using a standard curve of recombinant proteins to normalize signals across species. For tissue analysis, optimize fixation protocols for each species separately, as fixation efficiency can vary significantly. Implement multiplexed immunofluorescence with species-specific markers to distinguish target protein localization patterns across organisms. This methodological approach has proven effective in studies with antibodies like anti-amyloid beta [6E10], which has been validated for detecting amyloid in various sample types across species . Finally, verify findings with orthogonal methods such as RNA-seq to confirm that observed protein differences reflect biological variation rather than antibody affinity differences.

How can advanced imaging techniques enhance AEE10 antibody-based research?

Advanced imaging techniques can significantly enhance AEE10 antibody-based research by providing spatial, temporal, and quantitative insights into target protein biology. Implement super-resolution microscopy (such as STED or PALM) to visualize subcellular localization with precision below the diffraction limit, revealing potential compartmentalization of acyl-CoA synthetase activity. For dynamic studies, combine AEE10 antibody with FRAP (Fluorescence Recovery After Photobleaching) to measure protein mobility and residence time at different cellular locations. Live-cell imaging with a fluorescently tagged secondary antibody fragment can track protein redistribution during metabolic challenges. Quantitative image analysis should include colocalization studies with organelle markers and intensity correlation analysis. This multi-dimensional imaging approach has proven valuable in studies with other specialized antibodies, such as those used to investigate viral infections, where immunofluorescence analysis revealed distinct patterns between treated and untreated cells . Additionally, implement multiplexed imaging using spectral unmixing to simultaneously visualize AEE10 target protein alongside interaction partners or pathway components.

What are the common causes of false positives or background issues with AEE10 antibody, and how can they be addressed?

Common causes of false positives or high background with AEE10 antibody include insufficient blocking, too high primary or secondary antibody concentration, cross-reactivity with similar proteins, and inadequate washing. To systematically address these issues, implement a methodological troubleshooting protocol. First, optimize blocking by testing different blocking agents (BSA, milk, commercial blockers) and concentrations (1-5%). Perform titration experiments with primary antibody concentrations ranging from 0.1-5 μg/ml to identify the optimal signal-to-noise ratio . For improved specificity, increase wash duration and volume, using at least three 10-minute washes with gentle agitation. Consider using specialized low-background detection systems similar to those used with THE™ Phosphotyrosine Antibody (E10)plus, which demonstrated clear signal differentiation between stimulated and unstimulated samples . To address potential cross-reactivity, perform pre-adsorption controls by incubating the antibody with excess purified target protein before application. Finally, include multiple negative controls lacking the target protein but containing potential cross-reactive species to identify and characterize any non-specific binding.

How should researchers approach quantitative analysis of Western blot data generated with AEE10 antibody?

For quantitative analysis of Western blot data using AEE10 antibody, researchers should implement a comprehensive methodological framework. Begin by ensuring linear range detection by running a dilution series of your sample to confirm that the signal intensity proportionally corresponds to protein amount. Use appropriate housekeeping proteins for normalization, selecting those that remain stable under your experimental conditions. Employ dedicated image analysis software that can perform background subtraction and accurately define signal boundaries. For comparing multiple blots, include a common calibrator sample on each membrane and normalize all measurements to this reference. Statistical analysis should include at least three biological replicates, with appropriate statistical tests based on data distribution. This quantitative approach aligns with methodologies used for other specialized antibodies, such as anti-EphA10 mAbs, where comparative analysis between different samples revealed significant differences in expression patterns . Additionally, consider using fluorescently-labeled secondary antibodies rather than chemiluminescence for more accurate quantification, as they typically provide a broader linear dynamic range.

What strategies are recommended for validating antibody specificity when using AEE10 antibody in diverse experimental systems?

To validate AEE10 antibody specificity across diverse experimental systems, implement a multi-faceted validation strategy. Begin with genetic validation using knockout/knockdown models where the target protein is eliminated or reduced, confirming that antibody signal diminishes accordingly. For systems lacking genetic tools, perform immunodepletion experiments where the antibody is pre-incubated with purified target protein before application to samples. This should abolish specific signals, as demonstrated in validation studies for other research antibodies . Implement peptide competition assays using synthesized peptides corresponding to the antibody epitope, which should competitively reduce specific binding. Cross-validate findings using orthogonal methods such as mass spectrometry to confirm the identity of immunoprecipitated proteins. For tissue or cell type-specific validation, use RNA-sequencing data to correlate protein detection with mRNA expression patterns. Additionally, compare reactivity patterns across multiple antibodies targeting different epitopes of the same protein, similar to the comparison of phosphotyrosine antibodies (5E10, 18E10, E10 plus) that revealed distinct but overlapping recognition patterns .

How might AEE10 antibody contribute to understanding plant metabolic adaptations to environmental stress?

AEE10 antibody offers a valuable tool for investigating plant metabolic adaptations to environmental stress through its ability to detect changes in acyl-CoA synthetase expression and activity. Researchers can design time-course experiments exposing plants to different stressors (drought, salinity, temperature extremes) followed by protein extraction and Western blot analysis using AEE10 antibody to quantify target protein levels. Immunohistochemistry with AEE10 antibody on plant tissue sections can reveal tissue-specific regulation of the target enzyme during stress responses. By combining AEE10 antibody-based detection with metabolomic analysis of fatty acid profiles, researchers can establish correlations between enzyme expression and metabolic outcomes. For mechanistic insights, chromatin immunoprecipitation (ChIP) experiments can identify transcription factors regulating AEE10 target gene expression during stress. This multi-dimensional approach mirrors successful strategies employed with other specialized antibodies, such as those used to study virus-host interactions, where combining antibody-based detection with functional assays revealed mechanistic insights into antiviral activities .

What are the methodological approaches for using AEE10 antibody in protein-protein interaction studies?

For protein-protein interaction studies using AEE10 antibody, researchers should implement a multi-method approach to ensure robust findings. Begin with co-immunoprecipitation experiments where AEE10 antibody is used to pull down the target protein along with its interaction partners from cell lysates, followed by mass spectrometry analysis to identify these partners. To verify direct interactions, employ proximity ligation assays (PLA) which generate fluorescent signals only when two proteins are in close proximity (≤40 nm). For visualizing interactions in living cells, consider adapting bimolecular fluorescence complementation (BiFC) by conjugating complementary fluorescent protein fragments to AEE10 antibody fragments and potential interaction partners. Formal validation should include reciprocal co-immunoprecipitation, competition assays with blocking peptides, and domain mapping to identify specific interaction interfaces. Similar methodological approaches have been successfully implemented with antibodies like anti-EphA10 mAbs, where interaction studies revealed important insights into receptor biology and downstream signaling . Additionally, implement controlled analyses using both native and denaturing conditions to distinguish between direct and indirect interactions within larger protein complexes.

What novel applications of AEE10 antibody could emerge from combining traditional immunodetection with emerging technologies?

The integration of AEE10 antibody with emerging technologies presents exciting opportunities for novel research applications. Combining AEE10 antibody with CRISPR-Cas9 gene editing allows for precise correlation between genetic modifications and protein expression patterns. Researchers can implement antibody-guided chromatin profiling to investigate how the target protein associates with specific genomic regions, providing insights into potential regulatory functions beyond its enzymatic activity. Single-cell proteomics using AEE10 antibody-based detection can reveal heterogeneity in target protein expression across cell populations, particularly valuable for developmental studies. The application of AEE10 antibody in microfluidic devices enables high-throughput screening of compounds affecting target protein levels or activity. Similar innovative approaches have emerged with other specialized antibodies, such as engineered bispecific antibodies that demonstrate enhanced potency and breadth in neutralizing target antigens . Additionally, researchers could develop AEE10 antibody-based biosensors by conjugating the antibody to nanomaterials or fluorescent reporters that change properties upon target binding, allowing real-time monitoring of target protein dynamics in various experimental systems.