At3g59930 Antibody

What is the At3g59930 antibody and what does it specifically target?

At3g59930 antibody refers to monoclonal antibodies generated against Arabidopsis thaliana Actin-7, a protein encoded by the At3g59930 gene. These antibodies specifically recognize A. thaliana Actin-7, which plays crucial roles in plant growth, development, and response to environmental stimuli. Actin-7 has been identified in plants, animals, and protists, with the Arabidopsis variant being particularly important in auxin-mediated developmental processes, cell morphology changes, and cytoarchitecture formation. The protein is essential for callus tissue formation, germination, and root growth, and is expressed in rapidly developing tissues responsive to external stimuli such as hormonal exposure .

What are the primary applications for At3g59930 antibodies in plant research?

At3g59930 antibodies are valuable tools for investigating Actin-7's role in plant development and stress responses. According to available data, these antibodies have been validated for Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF) applications. Researchers commonly employ these antibodies to study cytoskeletal dynamics during plant growth, examine actin reorganization in response to hormones like auxin, investigate cellular morphogenesis processes, and analyze plant stress responses where the actin cytoskeleton undergoes significant remodeling. It is advisable to use all three monoclonal antibody clones (29G12.G5.G6, 33E8.C11.F5.D1, 36H8.C12.H10.B6) in initial qualitative experimental setups to determine which is most suitable for a specific experiment .

How is the At3g59930 antibody generated and purified?

The At3g59930 antibodies are mouse monoclonal antibodies (IgG isotype) developed by immunizing BALB/c mice with A. thaliana Actin-7 as the immunogen. The production process involves specialized hybridoma technology that yields stable antibody-producing cell lines. Following production, these antibodies undergo purification using Protein G affinity chromatography, which exploits the high binding affinity between Protein G and the Fc region of IgG antibodies. This purification method ensures high specificity and minimal cross-reactivity in experimental applications. The purified antibodies are formulated in phosphate-buffered saline (PBS) containing 0.05% (w/v) sodium azide as a preservative .

How should I validate the specificity of At3g59930 antibody in my experimental system?

Validating antibody specificity is crucial for reliable experimental results. For At3g59930 antibody, begin with Western blot analysis using both wild-type Arabidopsis extracts and actin-7 knockout/knockdown lines to confirm specific binding to the target protein (approximately 42 kDa). Include positive controls (purified recombinant Actin-7) and negative controls (non-plant actin or samples from species with divergent actin sequences). Perform immunoprecipitation followed by mass spectrometry to confirm that the antibody is pulling down the correct protein. For immunofluorescence applications, compare staining patterns with other known actin markers and perform competition assays with purified antigen. When publishing, document which of the three available monoclonal antibody clones (29G12.G5.G6, 33E8.C11.F5.D1, or 36H8.C12.H10.B6) provided optimal results for your specific application .

How can I differentiate between Actin-7 and other actin isoforms when using this antibody?

Differentiating between actin isoforms presents a significant challenge due to their high sequence homology. At3g59930 antibodies were specifically generated against Arabidopsis thaliana Actin-7, but potential cross-reactivity with other actin isoforms should be carefully evaluated. Implement a multi-faceted approach combining: (1) Preliminary Western blot analysis comparing wild-type and actin-7 mutant plants to confirm specificity, (2) Pre-absorption controls where the antibody is pre-incubated with purified Actin-7 before use, which should eliminate specific signals, (3) Two-dimensional gel electrophoresis followed by Western blotting to separate actin isoforms based on both molecular weight and isoelectric point before immunodetection, and (4) Complementary molecular techniques such as isoform-specific RT-PCR to correlate protein detection with transcript levels. Consider using the antibody in conjunction with the mAbGEa universal anti-actins antibody to identify the full actin complement versus the specific Actin-7 isoform .

What are the optimal conditions for using At3g59930 antibody in immunoprecipitation experiments?

For optimal immunoprecipitation (IP) of Actin-7 using At3g59930 antibody, implement a specialized protocol designed for cytoskeletal proteins. Begin with fresh plant tissue (preferably 0.5-1g) and homogenize in a cytoskeleton-stabilizing extraction buffer containing 50mM PIPES (pH 6.9), 5mM MgSO₄, 5mM EGTA, 5% glycerol, 0.1% Nonidet P-40, with freshly added 1mM DTT, 1mM PMSF, and protease inhibitor cocktail. After homogenization, centrifuge at 16,000×g for 15 minutes at 4°C. Pre-clear the supernatant with 50μl Protein G beads for 1 hour at 4°C. Incubate the pre-cleared lysate with 5-10μg of At3g59930 antibody overnight at 4°C with gentle rotation. Add 50μl fresh Protein G beads and incubate for 3 hours at 4°C. Wash beads 5 times with extraction buffer and once with PBS. Elute bound proteins with 50μl of 2× SDS sample buffer at 95°C for 5 minutes. This protocol maintains actin structure during extraction and optimizes antibody-antigen interaction during the immunoprecipitation process .

How can I adapt GPCR antibody discovery methods to improve At3g59930 antibody performance?

Recent advances in G protein-coupled receptor (GPCR) antibody discovery platforms offer innovative approaches that can be adapted to enhance At3g59930 antibody performance. Implementing stabilization strategies similar to those used for membrane proteins can improve antibody-antigen interactions. Consider employing amphiphilic poly-γ-glutamate (APG) technology, particularly APG-O which has shown success in stabilizing difficult-to-express proteins while maintaining their native conformation. This approach could be especially valuable when working with membrane-associated actin populations or when studying actin-membrane protein interactions. For developing next-generation At3g59930 antibodies with improved specificity, apply phage display technology using a three-phase selection strategy: (1) positive selection against Actin-7, (2) negative selection against other actin isoforms, and (3) final positive selection under native conditions. This approach, similar to that used for GPCRs, can yield antibodies with exceptional specificity even for closely related protein isoforms .

What structural considerations are important when using At3g59930 antibody to study actin-protein interactions?

When investigating actin-protein interactions using At3g59930 antibody, several structural considerations must be addressed. First, determine whether the antibody's epitope overlaps with binding sites of Actin-7 interaction partners by performing epitope mapping through peptide arrays or hydrogen-deuterium exchange mass spectrometry. This information is crucial to prevent antibody binding from disrupting or artificially enhancing protein interactions. Second, optimize fixation protocols that preserve both actin filaments and associated proteins - a modified dual fixation approach using 0.5% glutaraldehyde followed by 4% paraformaldehyde often provides the best results. Third, when performing co-immunoprecipitation studies, use reversible crosslinking agents like DSP (dithiobis(succinimidyl propionate)) to capture transient interactions before antibody application. Finally, complement antibody-based approaches with proximity ligation assays (PLA) to visualize and quantify interactions in situ with nanometer resolution. This integrated approach provides both qualitative and quantitative data about Actin-7's interaction network while mitigating potential artifacts introduced by the antibody itself .

How can I address non-specific binding issues when using At3g59930 antibody?

Non-specific binding is a common challenge when working with antibodies against highly conserved proteins like actins. To address this issue with At3g59930 antibody, implement a systematic optimization strategy. First, increase blocking stringency by using 5% BSA or 5% milk with 0.2% Tween-20 in TBS for Western blots or 3% BSA with 0.3% Triton X-100 for immunofluorescence. Second, optimize antibody dilution by performing a dilution series (1:500 to 1:5000) to identify the concentration that maximizes specific signal while minimizing background. Third, include competitive inhibitors such as 0.1-0.2% poly-L-lysine in your antibody diluent to reduce charge-based non-specific interactions. Fourth, for plant tissues with high autofluorescence or phenolic compounds, pre-incubate sections with 0.1M glycine followed by 0.5M ammonium chloride to reduce background. Finally, consider using size-exclusion chromatography to further purify the antibody before use, as this can remove potential aggregates that contribute to non-specific binding. Document which combination of these approaches works best for your specific plant tissue and experimental conditions .

What strategies can resolve contradictory results between different detection methods using At3g59930 antibody?

When encountering contradictory results between different detection methods (e.g., Western blot versus immunofluorescence) using At3g59930 antibody, implement a structured analytical approach. Begin by evaluating method-specific variables: for Western blots, try different extraction buffers, denaturation conditions, and transfer parameters; for immunofluorescence, test multiple fixation protocols and antigen retrieval methods. Next, perform a cross-validation study using all three available monoclonal antibody clones (29G12.G5.G6, 33E8.C11.F5.D1, 36H8.C12.H10.B6) in parallel across all methods to determine if discrepancies are antibody-specific. Consider epitope accessibility issues - certain detection methods may expose different regions of the protein. Implement alternative detection strategies such as proximity ligation assay or immunoelectron microscopy to provide additional data points. Finally, correlate results with orthogonal techniques such as fluorescently tagged Actin-7 constructs or mass spectrometry-based protein identification. This comprehensive approach not only resolves contradictions but often yields deeper insights into protein behavior across different experimental contexts .

How can computational approaches from antibody design be applied to optimize At3g59930 antibody experimental protocols?

Computational approaches from modern antibody design can significantly enhance experimental protocols using At3g59930 antibody. Implement sequence-based prediction models similar to DyAb to optimize buffer conditions and experimental parameters. First, use antibody sequence analysis to predict physicochemical properties that influence performance in different applications. Second, apply machine learning algorithms trained on antibody-antigen interaction data to predict optimal binding conditions - including pH, salt concentration, and detergent composition for your specific experimental system. Third, utilize protein structure prediction to model the interaction between At3g59930 antibody and Actin-7, identifying potential binding constraints or conformational requirements. Fourth, employ regression models similar to those used in therapeutic antibody development to predict how protocol modifications might affect experimental outcomes. Finally, implement genetic algorithm approaches to systematically optimize multiple experimental parameters simultaneously, similar to the methodology used in affinity maturation studies. This computational-experimental hybrid approach can dramatically reduce optimization time while improving reproducibility and sensitivity .

What are the detailed technical specifications of available At3g59930 antibodies?

The technical specifications of commercially available At3g59930 antibodies are summarized in the following table:

These antibodies were developed in the laboratory of Richard B. Meagher, PhD, University of Georgia. It is advisable to use all three monoclonal antibodies in first-time qualitative experimental setups to determine which is most suitable for specific experimental conditions. These antibodies may be used to complement experiments carried out with mAbGEa universal anti-actins antibody for comparative analyses .

How does the performance of At3g59930 antibody compare with other plant actin antibodies?

Comparative analysis of At3g59930 antibody performance against other plant actin antibodies reveals distinct advantages and limitations. Unlike pan-actin antibodies (such as mAbGEa), At3g59930 antibodies offer isoform-specific detection, allowing researchers to distinguish Actin-7's unique roles from other actin proteins. In Western blot applications, At3g59930 antibodies typically demonstrate higher specificity but potentially lower sensitivity compared to commercial pan-actin antibodies. For immunofluorescence, At3g59930 antibodies excel in developmental studies where precise localization of Actin-7 is required, but may require more stringent optimization in tissues with complex matrices. When comparing reproducibility across different experimental batches, monoclonal At3g59930 antibodies show superior consistency compared to polyclonal alternatives, though with potentially narrower epitope recognition. For co-immunoprecipitation studies, At3g59930 antibodies demonstrate excellent performance for Actin-7-specific interaction studies but may not capture the complete actin interactome. This comparative performance profile highlights the importance of selecting the appropriate antibody based on specific experimental goals - use At3g59930 antibodies when isoform discrimination is critical, and pan-actin antibodies when detecting the general actin cytoskeleton .

How can genetic algorithm approaches improve At3g59930 antibody design for challenging research applications?

Genetic algorithm (GA) approaches offer promising avenues for optimizing At3g59930 antibody design, particularly for challenging research applications like in situ protein-protein interaction studies. Implementing a GA-based optimization strategy similar to that described for therapeutic antibodies could dramatically improve antibody performance. The process would begin with generating a diverse library of antibody variants through CDR modifications, followed by iterative rounds of selection based on predefined fitness parameters (specificity, sensitivity, and stability in plant extracts). This approach could be particularly valuable for developing antibody variants optimized for different fixation conditions or detection methods. For example, a GA could identify antibody variants with enhanced performance in formaldehyde-fixed tissues or improved stability in extracts containing plant-specific compounds. The workflow would involve: (1) starting with the parental clone sequences for existing At3g59930 antibodies, (2) identifying mutations that individually improve binding characteristics, (3) combining 3-4 beneficial mutations to generate new sequence variants, (4) scoring these variants with computational prediction models, and (5) selecting the most promising candidates for experimental validation. This iterative approach could yield specialized antibody variants optimized for specific plant tissues or experimental conditions .

What novel applications could emerge from combining At3g59930 antibody with advanced imaging technologies?

The integration of At3g59930 antibody with emerging advanced imaging technologies presents exciting opportunities for novel research applications. Combining this antibody with super-resolution microscopy techniques such as STORM (Stochastic Optical Reconstruction Microscopy) or PALM (Photoactivated Localization Microscopy) could reveal unprecedented details of Actin-7 organization within plant cells at nanometer resolution. Implementation of expansion microscopy protocols with At3g59930 antibody would allow physical expansion of plant tissue samples while maintaining antibody labeling, effectively improving resolution on standard confocal microscopes. Lattice light-sheet microscopy coupled with At3g59930 immunolabeling could enable real-time visualization of Actin-7 dynamics in live plant cells with minimal phototoxicity. Correlative light and electron microscopy (CLEM) approaches would bridge the resolution gap between immunofluorescence and ultrastructural analysis. Perhaps most promising is the combination of At3g59930 antibody with multiplexed ion beam imaging (MIBI) or imaging mass cytometry (IMC), which would allow simultaneous detection of Actin-7 alongside dozens of other proteins, providing comprehensive spatial proteomics data in plant tissues. These technological integrations could transform our understanding of Actin-7's role in plant development and stress responses .

How might sequence-based antibody design platforms improve At3g59930 antibody development for plant research?

Sequence-based antibody design platforms like DyAb represent a transformative approach for developing next-generation At3g59930 antibodies with enhanced performance characteristics. These computational platforms could address several key challenges in plant antibody research. First, by analyzing sequence-structure-function relationships, designers could develop antibody variants with improved specificity for distinguishing between highly similar actin isoforms. Second, stability-enhancing modifications could be identified to improve antibody performance in plant-specific extraction buffers containing specialized detergents and stabilizing agents. Third, the relative embedding approach used in DyAb could be adapted to predict antibody performance across diverse plant species, facilitating cross-species studies. For implementation, researchers would start with a small dataset (~100 variants) of existing At3g59930 antibodies with measured binding affinities, train a DyAb-like model on these data, and use the model to design novel variants with improved properties. This approach is particularly valuable given the difficulty of obtaining large labeled datasets in specialized research fields like plant cytoskeleton studies. The result would be antibodies specifically optimized for plant research applications, potentially achieving 3-10 fold improvements in affinity and specificity compared to current versions .