At2g19940 Antibody

What is AT2G19940 and why would researchers develop antibodies against it?

AT2G19940 is a protein-coding gene in Arabidopsis thaliana located on chromosome 2, annotated as a putative N-acetyl-gamma-glutamyl-phosphate reductase . According to functional annotation, this protein is involved in oxidoreductase activity, acting on aldehyde or oxo groups of donors, with NAD or NADP as acceptors, and also possesses copper ion binding properties . Researchers develop antibodies against AT2G19940 to study its expression patterns, protein localization, protein-protein interactions, and functional roles in metabolic pathways involving oxidoreductase activity.

What types of antibodies are most suitable for detecting AT2G19940 protein?

For AT2G19940 research, both polyclonal and monoclonal antibodies have specific applications:

Polyclonal antibodies, like those developed against other Arabidopsis proteins, are typically generated using synthetic peptides derived from unique regions of AT2G19940 conjugated to carrier proteins like KLH (keyhole limpet hemocyanin) . For optimal results, immunogen affinity-purified antibodies in PBS (pH 7.4) provide the best balance of specificity and sensitivity when working with plant proteins.

How can researchers obtain reliable antibodies for AT2G19940?

Given the specialized nature of plant research antibodies, researchers can:

• Commission custom antibody production using synthetic peptides derived from AT2G19940 sequence

• Utilize trial programs from specialized plant antibody providers that offer initial testing quantities (20 μg/μL) for validation before larger investments

• Check plant-specific antibody repositories that maintain validated antibodies for Arabidopsis research

• Develop in-house antibodies following established protocols for plant protein immunization

When selecting commercial options, verify that providers have experience with plant proteins, as antibodies optimized for mammalian systems may have reduced efficacy in plant research.

What are the optimal extraction methods when preparing AT2G19940 protein samples for antibody-based detection?

For effective AT2G19940 protein extraction from Arabidopsis tissues:

Based on experimental procedures used for similar Arabidopsis proteins, TCA-acetone precipitation followed by solubilization in urea buffer has proven effective for extracting membrane-associated and enzymatic proteins . For AT2G19940, which may be involved in metabolic pathways, adding protease inhibitors is crucial to prevent degradation during extraction.

What are the recommended protocols for Western blot detection of AT2G19940?

For optimal Western blot detection of AT2G19940:

• Separate 300 μg of total protein extract on 15% SDS-PAGE (1 mm thick gel)

• Transfer to 0.2 μm nitrocellulose membrane at 100V for 1 hour using wet transfer system

• Block with 0.5% cold fish gelatin in TBS for 1 hour at room temperature

• Incubate with primary antibody at 1:250-1:500 dilution for 1 hour at room temperature

• Wash 3× with TBS-TT (15 minutes each)

• Incubate with secondary antibody (anti-rabbit/anti-chicken conjugated to fluorophore or HRP) at 1:5000 dilution

• Perform final washes and visualization according to detection system

Based on the expected molecular weight of AT2G19940 (similar to other N-acetyl-gamma-glutamyl-phosphate reductases), researchers should look for a band at approximately 38-45 kDa, though post-translational modifications may alter migration patterns.

How can researchers validate the specificity of AT2G19940 antibodies?

Comprehensive validation strategies include:

• Genetic Controls: Compare wild-type plants with knockout/knockdown mutants (if available) for AT2G19940. Similar approaches used for Arabidopsis AGO2 showed complete absence of signal in knockout lines .

• Peptide Competition Assay: Pre-incubate antibody with excess immunogenic peptide before application to samples, which should eliminate specific binding.

• Recombinant Protein Controls: Express and purify recombinant AT2G19940 protein for positive control and antibody titration.

• Cross-Reactivity Testing: Test against related Arabidopsis proteins to confirm specificity, particularly other oxidoreductases or NAD/NADP-binding proteins.

• Multiple Detection Techniques: Confirm results across different methods (Western blot, immunoprecipitation, immunofluorescence) to build confidence in antibody specificity.

What advanced affinity maturation strategies could improve AT2G19940 antibody performance?

For researchers seeking to optimize AT2G19940 antibody performance, advanced affinity maturation techniques can significantly enhance binding properties and specificity:

• Shuffle/ShuffleStEP Method: This unbiased optimization approach combines antibody chain shuffling with staggered-extension processes to recombine beneficial mutations from all six complementarity-determining regions (CDRs), significantly improving binding affinity and inhibitory potency .

• Pool Maturation: Simultaneously optimize multiple lead antibody candidates to identify superior variants with desired properties, as demonstrated in Arginase 2 inhibitory antibody development .

• Diverse Library Construction: Generate broad sequence diversity to increase the structural repertoire from which superior binding variants can be selected, overcoming limitations of conventional strategies that focus on small antibody regions .

Implementing these advanced techniques can achieve substantial improvements in AT2G19940 antibody binding properties, particularly important when studying low-abundance plant proteins in complex tissue samples.

How can researchers address potential artifacts when using AT2G19940 antibodies in Arabidopsis tissues?

When using antibodies against AT2G19940 in Arabidopsis tissues, researchers should be aware of potential artifacts and implement appropriate controls:

• Endogenous Biotin Interference: Plant tissues contain high levels of endogenous biotin that can cause background with biotin-streptavidin detection systems. Use alternative detection methods or specific blocking steps to mitigate this issue.

• Plant Secondary Metabolites: Phenolic compounds and other plant metabolites can interfere with antibody binding. Include appropriate sample preparation steps (PVPP treatment, reducing agents) to minimize these effects.

• Cross-Reactivity with Related Proteins: AT2G19940's function as an oxidoreductase suggests potential structural similarity with other NAD/NADP-binding proteins. Validate antibody specificity against related Arabidopsis proteins.

• Tissue-Specific Expression Variability: Consider that AT2G19940 expression may vary significantly across tissues and developmental stages. Include appropriate positive controls from tissues with known expression.

• Fixation Artifacts: When performing immunohistochemistry, different fixation methods can affect epitope accessibility. Optimize fixation protocols specifically for AT2G19940 detection.

How can AT2G19940 antibodies be utilized in studying stress response pathways in Arabidopsis?

AT2G19940 antibodies can elucidate protein involvement in stress response through:

• Protein Level Monitoring: Track AT2G19940 protein levels under various stress conditions (drought, heat, salt) through quantitative Western blot analysis.

• Subcellular Localization Changes: Use immunofluorescence to monitor potential stress-induced relocalization of AT2G19940.

• Protein-Protein Interaction Studies: Employ co-immunoprecipitation with AT2G19940 antibodies to identify interacting partners during stress response.

• Post-Translational Modification Analysis: Combine AT2G19940 immunoprecipitation with mass spectrometry to identify stress-induced modifications.

• ChIP Analysis: If AT2G19940 has any DNA-binding properties, chromatin immunoprecipitation can reveal genome interaction sites.

For experimental design, researchers should include time-course analyses of multiple stress treatments, comparing protein dynamics with transcriptional changes identified through microarray studies similar to those conducted for recombinant antibody expression .

What considerations should researchers address when using AT2G19940 antibodies in transgenic Arabidopsis lines?

When working with transgenic Arabidopsis lines and AT2G19940 antibodies:

• Unfolded Protein Response (UPR) Effects: Overexpression of proteins can trigger endoplasmic reticulum stress and the unfolded protein response, potentially affecting native AT2G19940 expression. Monitor UPR markers as demonstrated in antibody-expressing Arabidopsis seeds, where microarray analysis identified 27 consistently upregulated genes across transgenic lines .

• Tag Interference: If studying tagged versions of AT2G19940, validate that the tag doesn't interfere with antibody binding or protein function.

• Expression Level Variation: Account for position effects in transgenic lines causing variable expression levels. Use multiple independent lines for confirmation.

• Developmental Timing: Consider developmental regulation when designing experiments, as protein expression patterns may vary significantly throughout the plant life cycle.

• Background Genotype Effects: Different Arabidopsis ecotypes may show variation in AT2G19940 expression or function. Maintain consistent genetic backgrounds when making comparisons.

What emerging technologies could enhance AT2G19940 antibody applications in plant research?

Cutting-edge approaches for AT2G19940 research include:

• Proximity Labeling: Combine AT2G19940 antibodies with TurboID or APEX2 proximity labeling to identify transient interaction partners and neighboring proteins in the native cellular environment.

• Single-Cell Proteomics: Adapt AT2G19940 antibodies for use in emerging plant single-cell proteomic techniques to understand cell-type-specific expression patterns.

• Spatial Transcriptomics Integration: Correlate immunohistochemistry results with spatial transcriptomics data to build comprehensive models of AT2G19940 regulation across tissue types.

• Nanobody Development: Design AT2G19940-specific nanobodies for applications requiring smaller binding agents, such as super-resolution microscopy or intracellular targeting.

• CRISPR-Epitope Tagging: Use CRISPR-Cas9 to introduce epitope tags at the endogenous AT2G19940 locus, allowing antibody detection while maintaining native expression patterns and regulation.