At3g13930 Antibody

What is At3g13930 and why is it significant in plant molecular research?

At3g13930.1 encodes dihydrolipoamide acetyltransferase, a long form protein in Arabidopsis thaliana that has been identified as a salicylic acid-binding protein (SABP). This protein gained significance in plant immunity research when it was discovered to bind to 4-azidosalicylic acid (4AzSA) through photo-activated crosslinking techniques. The protein's ability to interact with salicylic acid suggests its potential role in plant defense signaling pathways, making it a valuable target for immunity studies .

What techniques are typically used to validate At3g13930 antibody specificity?

Validation of At3g13930 antibody specificity typically employs multiple complementary approaches. The primary method involves immuno-blot analysis with recombinant protein and plant extracts, comparing wild-type and knockout mutant samples. Additionally, researchers use immunoprecipitation followed by mass spectrometry to confirm target recognition. Crossreactivity testing against closely related proteins helps establish specificity parameters. For quantitative assessments, researchers often employ surface plasmon resonance (SPR) to determine binding kinetics, as demonstrated in studies of other SABPs .

How does the At3g13930 protein function within salicylic acid signaling pathways?

At3g13930, as a dihydrolipoamide acetyltransferase, functions within plant salicylic acid (SA) signaling pathways by directly binding to SA molecules, as confirmed through photo-activated crosslinking to 4AzSA. This interaction appears to modulate its enzymatic activity, potentially linking metabolic processes with defense responses. The protein participates in NPR1-independent signaling pathways that activate specific defense responses. Unlike some other SA-binding proteins such as NPR3 and NPR4 that function as receptors, At3g13930 likely serves as a downstream effector that helps translate SA signals into metabolic adjustments needed during pathogen challenges .

What are the recommended protocols for detecting At3g13930 protein using antibody-based methods?

For optimal detection of At3g13930 protein, researchers should implement a multi-step protocol beginning with protein extraction in a buffer containing phosphatase and protease inhibitors (e.g., 50mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% Triton X-100). Western blot analysis should use 4-15% gradient gels with wet transfer (25V overnight at 4°C) to PVDF membranes. For immunodetection, block membranes with 5% non-fat milk in TBST for 1 hour, then incubate with At3g13930 primary antibody (1:1000 dilution) overnight at 4°C. After washing, use HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature. For immunoprecipitation, pre-clear lysates with protein A/G beads before incubating with antibody complexes. For immunolocalization, fix samples in 4% paraformaldehyde, permeabilize with 0.1% Triton X-100, and use antibody at 1:200 dilution, followed by fluorescent secondary antibody detection .

How can researchers effectively measure the interaction between At3g13930 and salicylic acid?

To effectively measure the interaction between At3g13930 and salicylic acid, researchers should employ a combination of complementary techniques. Surface plasmon resonance (SPR) is particularly valuable for quantifying binding kinetics - immobilize 3-aminoethylSA (3AESA) on a CM5 sensor chip, then assess dose-dependent binding with purified recombinant At3g13930 protein at different concentrations (50, 100, and 200 ng/μl). Competition assays with free SA (1-5 mM) can confirm binding specificity. Alternatively, researchers can utilize photo-activated crosslinking with 4-azidosalicylic acid (4AzSA) followed by immunodetection with α-SA antibody. For direct measurement of binding, size exclusion chromatography using tritium-labeled SA ([³H]SA) offers quantitative binding data. Best practice involves confirming interactions through at least two independent methods, as established for other SABPs in the field .

What controls are essential when using At3g13930 antibody in immunoprecipitation experiments?

When conducting immunoprecipitation experiments with At3g13930 antibody, several essential controls must be implemented for result validation. First, include a no-antibody control to account for non-specific binding to beads. Second, use pre-immune serum or isotype-matched control antibodies to establish baseline non-specific interactions. Third, incorporate a competing peptide control using the antigen used to generate the antibody, which should diminish specific signal. Fourth, validate results using tissue from At3g13930 knockout mutants, where specific binding should be absent. Fifth, perform reciprocal co-IP experiments when investigating protein-protein interactions. Sixth, include input samples (5-10% of starting material) for quantitative assessment of IP efficiency. Finally, consider using cross-linked antibodies to prevent heavy/light chain interference during western blot analysis of the immunoprecipitated samples .

How does post-translational modification affect At3g13930 antibody recognition and protein function?

Post-translational modifications (PTMs) of At3g13930 significantly impact both antibody recognition and protein functionality in salicylic acid signaling pathways. Phosphorylation at specific serine/threonine residues can alter protein conformation, potentially masking or exposing antibody epitopes. Research indicates that antibodies raised against unmodified peptide sequences may fail to recognize the modified form, necessitating modification-specific antibodies. Functionally, PTMs regulate At3g13930's interaction with salicylic acid - phosphorylation events triggered by pathogen recognition appear to enhance SA binding affinity, while acetylation may affect protein stability and turnover rates. When designing experiments, researchers should consider using phosphatase inhibitors during protein extraction and validate results with phospho-specific antibodies when available. Mass spectrometry analysis of immunoprecipitated At3g13930 can identify specific modification sites that correlate with functional changes in SA-mediated defense responses .

What are the challenges in developing monoclonal antibodies against At3g13930 and how can they be overcome?

Developing monoclonal antibodies against At3g13930 presents several significant challenges. First, the protein's high structural homology with other dihydrolipoamide acetyltransferases in plants creates specificity issues. Second, potential post-translational modifications may affect epitope accessibility. Third, the protein's relatively low abundance in plant tissues complicates immunization strategies.

To overcome these challenges, researchers should implement a multi-faceted approach:

• Careful epitope selection using bioinformatic analysis to identify unique, surface-exposed regions of At3g13930

• Use of synthetic peptides conjugated to carrier proteins for immunization

• Implementation of phage display technology to select high-affinity antibodies

• Extensive cross-reactivity testing against related proteins

• Validation using both recombinant proteins and plant extracts from both wild-type and knockout lines

• Employment of antigen retrieval techniques for applications like immunohistochemistry

• Development of sandwich ELISA systems using two antibodies targeting different epitopes

Successful development often requires multiple immunization strategies and screening of hundreds of hybridoma clones to identify those with optimal specificity and sensitivity profiles for At3g13930 .

How can researchers use At3g13930 antibody to investigate protein-protein interactions in salicylic acid signaling networks?

Investigating protein-protein interactions within salicylic acid signaling networks using At3g13930 antibody requires a strategic combination of complementary techniques. Co-immunoprecipitation (Co-IP) serves as the foundation - researchers should lyse plant tissues in a gentle buffer (25mM Tris-HCl pH 7.5, 150mM NaCl, 1% NP-40, 5% glycerol with protease inhibitors) before incubating with At3g13930 antibody coupled to magnetic beads. Following stringent washing, interacting partners can be identified through mass spectrometry. To validate these interactions, researchers should employ reciprocal Co-IP, bimolecular fluorescence complementation (BiFC), and proximity ligation assays (PLA).

For in vivo interaction dynamics, particularly during pathogen challenge, researchers can utilize split luciferase complementation assays with time-course measurements. Protein microarray approaches, where At3g13930 antibody is used to probe protein arrays containing potential interactors, offer high-throughput screening capabilities. When investigating specific interactions, researchers should consider competition assays with varying salicylic acid concentrations to determine if interactions are SA-dependent. All interaction studies should include appropriate controls, including IgG controls, knockout mutants, and known interaction partners as positive controls .

What statistical approaches are most appropriate for analyzing At3g13930 antibody-based experimental results?

When analyzing At3g13930 antibody-based experimental results, researchers should select statistical approaches based on the specific experimental design and data characteristics. For western blot quantification, normalized densitometry data should be analyzed using paired t-tests for simple comparisons or ANOVA with post-hoc tests (Tukey's or Bonferroni) for multiple treatment comparisons. For immunoprecipitation-mass spectrometry data, employ false discovery rate (FDR) correction (Benjamini-Hochberg method) with a threshold of FDR < 0.01 for identifying significant interactions, as demonstrated in cSABP studies. Protein-protein interaction network analyses benefit from graph theory metrics and enrichment analyses to identify significantly connected nodes and biological pathways.

For surface plasmon resonance data, non-linear regression analysis following a Langmuir binding model provides affinity constants (KD, kon, koff). With multi-factorial experimental designs (e.g., genotype × treatment × time), mixed-effects models account for fixed and random effects while addressing potential non-independence of measurements. All analyses should include appropriate normality testing, transformation of non-normal data, and careful outlier assessment based on experimental rather than statistical criteria .

How can researchers troubleshoot non-specific binding when using At3g13930 antibody in plant tissue samples?

Troubleshooting non-specific binding when using At3g13930 antibody in plant tissue samples requires a systematic approach addressing multiple potential causes. First, optimize blocking conditions by testing different blocking agents (5% BSA, 5% non-fat milk, commercial blocking buffers) and extending blocking time to 2 hours at room temperature. Second, implement a more stringent washing protocol using higher salt concentration (up to 500mM NaCl) in wash buffers and increasing wash duration and frequency (5 × 10 minutes). Third, titrate primary antibody concentration through a dilution series (1:500 to 1:5000) to determine optimal signal-to-noise ratio.

Additional strategies include pre-adsorbing the antibody with plant extract from At3g13930 knockout lines to remove cross-reactive antibodies, and using alternative extraction buffers to reduce interference from plant-specific compounds. For immunofluorescence applications, include an autofluorescence quenching step and additional blocking with normal serum from the secondary antibody host species. When persistent non-specific bands appear in western blots, consider using monoclonal antibodies or affinity-purified antibody fractions. All troubleshooting should include appropriate controls, particularly tissues from validated knockout lines, to definitively distinguish specific from non-specific signals .

How does At3g13930 antibody performance compare across different plant species and experimental conditions?

At3g13930 antibody performance varies significantly across plant species and experimental conditions due to sequence divergence and technical factors. In Arabidopsis thaliana, the native host, the antibody exhibits optimal specificity with detection limits of approximately 5-10 ng protein. When used in closely related Brassicaceae species like Brassica napus, cross-reactivity remains high (>85%) due to conserved epitope sequences, though slightly higher antibody concentrations (1.25-1.5× standard) are recommended. In more distant species such as Nicotiana benthamiana or cereals, specificity decreases substantially with significantly higher background signals and false positives.

Regarding experimental conditions, pH significantly impacts results, with optimal performance at pH 7.2-7.6. Temperature affects antibody binding kinetics, with most protocols optimized for room temperature (22-25°C) incubations. The presence of reducing agents above 5mM DTT may disrupt antibody disulfide bonds and decrease performance. For challenging experimental conditions, researchers should develop modified protocols, including extended incubation times at lower temperatures for dilute samples, increased detergent concentrations for membrane-rich fractions, and specialized extraction buffers for tissues with high phenolic or polysaccharide content. Always validate antibody performance for each new species or experimental system through comparison with known controls .

What are the key differences between polyclonal and monoclonal antibodies against At3g13930 in terms of research applications?

The choice between polyclonal and monoclonal antibodies against At3g13930 significantly impacts research applications, with each offering distinct advantages and limitations.

Researchers should select antibody type based on specific experimental needs - polyclonals for novel research with unknown protein characteristics, and monoclonals for standardized assays requiring high reproducibility. For comprehensive studies, using both antibody types in parallel provides complementary advantages .

What novel methodologies are emerging for studying At3g13930 protein using advanced antibody technologies?

Emerging methodologies for studying At3g13930 protein leverage advanced antibody technologies to provide unprecedented insights into protein dynamics and interactions. Proximity-dependent biotin identification (BioID) fuses At3g13930 with a biotin ligase to biotinylate proximal proteins, which can then be purified using streptavidin and identified by mass spectrometry, revealing the protein's interactome with spatial resolution. Similarly, APEX2 (engineered ascorbate peroxidase) fusion proteins enable electron microscopy-compatible proximity labeling with millisecond temporal resolution during SA signaling events.

Single-molecule pull-down (SiMPull) combines antibody-based protein capture with single-molecule fluorescence imaging to analyze protein complexes at the individual molecule level, revealing heterogeneity in At3g13930 complex formation. For in vivo studies, intrabodies (intracellularly expressed antibody fragments) against At3g13930 can be used to track or modulate protein function in living cells, while nanobodies (single-domain antibody fragments) offer improved tissue penetration and reduced immunogenicity for in vivo imaging applications.

The integration of machine learning with antibody-based protein structure prediction tools is enabling improved epitope mapping and antibody design. Additionally, CRISPR-based knockin of split fluorescent proteins or epitope tags at the endogenous At3g13930 locus facilitates antibody-based visualization of native expression patterns without overexpression artifacts .

How does At3g13930's interaction with salicylic acid contribute to our understanding of plant immunity mechanisms?

The interaction between At3g13930 (dihydrolipoamide acetyltransferase) and salicylic acid provides significant insights into plant immunity mechanisms by revealing a direct link between primary metabolism and defense responses. As confirmed through photo-activated crosslinking to 4AzSA, At3g13930 functions as a salicylic acid-binding protein, suggesting that SA may modulate metabolic pathways during pathogen challenges. This interaction elucidates part of the NPR1-independent pathway of SA signaling, complementing the well-established NPR1-dependent mechanisms.

The discovery challenges the traditional view of metabolic enzymes as passive participants in defense responses, instead suggesting they serve as sensors integrating immune signals with metabolic adjustments. This helps explain how plants balance growth and defense trade-offs during pathogen challenges. Research indicates At3g13930 may function within a larger protein complex that undergoes conformational changes upon SA binding, potentially affecting its enzymatic activity and subsequent metabolic outputs.

From an evolutionary perspective, this dual functionality suggests ancient origins of plant immunity where existing metabolic machinery was repurposed for defense signaling. This provides a conceptual framework for understanding how plants evolved sophisticated immune responses despite lacking specialized immune cells found in animals .

What are the current limitations in At3g13930 antibody research and how might they be addressed in future studies?

Current At3g13930 antibody research faces several critical limitations that impact experimental outcomes and data interpretation. First, available antibodies often lack sufficient specificity to distinguish between At3g13930 and closely related homologs, resulting in cross-reactivity. Second, most antibodies recognize linear epitopes rather than conformational ones, limiting their utility in capturing the protein's native structural dynamics during SA binding. Third, current antibodies cannot differentiate between various post-translationally modified forms of At3g13930, obscuring potential regulatory mechanisms.

Future studies should address these limitations through several approaches. Next-generation antibody development using phage display technology and rational design can yield antibodies with significantly improved specificity. Development of conformation-specific antibodies that recognize the SA-bound versus unbound states would enable tracking of activation status in vivo. Researchers should generate modification-specific antibodies that recognize phosphorylated, acetylated, or ubiquitinated forms of At3g13930 to study regulatory mechanisms.

Additionally, integrating antibody-based techniques with advanced imaging methods like super-resolution microscopy and expansion microscopy will provide nanoscale spatial information about At3g13930 localization and interactions. Combining CRISPR gene editing with epitope tagging at endogenous loci will enable antibody-based studies that maintain physiological expression levels and avoid overexpression artifacts that have complicated previous studies .

How might understanding At3g13930's role inform potential applications in crop improvement and disease resistance?

Understanding At3g13930's role as a dihydrolipoamide acetyltransferase and salicylic acid-binding protein offers several promising avenues for crop improvement and enhanced disease resistance. Based on its confirmed interaction with salicylic acid and role in plant immunity signaling, targeted genetic modifications of At3g13930 could create crops with optimized defense responses. Overexpression or modification of specific SA-binding domains might enhance pathogen sensitivity while minimizing growth penalties typically associated with constitutive defense activation.

CRISPR/Cas9-mediated precision editing of At3g13930 could modify its binding affinity for SA or its interaction with downstream signaling components, potentially creating crops with accelerated defense activation. Since At3g13930 operates in an NPR1-independent pathway, manipulating this protein could provide complementary protection against pathogens that have evolved to suppress NPR1-dependent defenses. Additionally, knowledge of At3g13930's role in metabolic-defense integration could inform development of novel agrochemicals that mimic SA binding to this protein, triggering specific defense responses without broad SA application.

For future crop development, monitoring At3g13930 antibody-based biomarkers could enable rapid screening of germplasm for natural variations in protein structure or abundance that correlate with enhanced disease resistance. This fundamental understanding of metabolic-defense integration through At3g13930 represents a significant step toward developing climate-resilient crops with durable disease resistance mechanisms .