At4g27220 Antibody

What is AT4G27220 and why is it significant in plant immunity research?

AT4G27220 encodes an NB-ARC domain-containing disease resistance protein in Arabidopsis thaliana, making it a member of the nucleotide-binding leucine-rich repeat (NLR) protein family . These proteins are central components of plant immune responses, functioning as intracellular receptors that recognize pathogen effectors and trigger defense mechanisms. The NB-ARC domain (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) is a conserved element in many plant resistance proteins that regulates protein activity through nucleotide binding and hydrolysis. Research on AT4G27220 contributes to our understanding of how plants detect and respond to pathogens, particularly in the context of disease resistance pathways and the complex regulatory networks of plant immunity.

How does AT4G27220 expression change during pathogen infection?

AT4G27220 expression is dynamically regulated during pathogen challenges. Transcriptomic analyses have shown that NLR genes, including AT4G27220, often undergo significant expression changes during infection events . Expression patterns may vary depending on the specific pathogen, infection stage, and plant tissue examined. In studies of mutants with altered resistance profiles (such as the adf4 mutant), AT4G27220 shows differential expression compared to wild-type plants, suggesting its involvement in actin cytoskeleton-mediated immune responses . When designing experiments to study AT4G27220, researchers should include appropriate time-course analyses to capture the temporal dynamics of expression changes and consider tissue-specific responses that might be overlooked in whole-plant analyses.

What are the main challenges in developing antibodies against plant NLR proteins like AT4G27220?

Developing antibodies against plant NLR proteins presents several challenges. First, many NLRs share high sequence homology, making it difficult to generate antibodies that specifically recognize AT4G27220 without cross-reactivity to related proteins. Second, NLRs are often expressed at relatively low levels in plants, requiring highly sensitive detection methods. Third, conformational changes in NLR proteins during activation can affect epitope accessibility, potentially limiting antibody recognition to specific protein states. When developing antibodies against AT4G27220, researchers should carefully select unique peptide regions as immunogens, validate specificity against related NLRs, and consider using a combination of monoclonal and polyclonal approaches to maximize detection capabilities across different experimental conditions.

What are the most effective strategies for generating specific antibodies against AT4G27220?

The most effective strategy for generating AT4G27220-specific antibodies involves careful antigen design to target unique regions of the protein. Begin by performing comprehensive sequence alignment with related Arabidopsis NLR proteins to identify regions with minimal homology to other proteins. The leucine-rich repeat (LRR) region often contains unique sequences suitable for antibody generation. Consider synthesizing peptides from multiple regions (15-25 amino acids each) to increase success probability. For recombinant protein antigens, express fragments rather than the full-length protein, as NB-ARC proteins can be difficult to express in soluble form. Compare KLH and BSA as carrier proteins, as some plant researchers report better results with KLH-conjugated peptides . Both rabbit polyclonal antibodies (for broader epitope recognition) and mouse monoclonal antibodies (for consistency across experiments) should be considered. Validate specificity using knockout lines (at4g27220 mutants) as negative controls and recombinant protein as a positive control.

How should researchers validate the specificity of antibodies targeting AT4G27220?

Comprehensive validation of AT4G27220 antibodies requires multiple complementary approaches. First, perform western blot analysis using proteins extracted from wild-type Arabidopsis and at4g27220 knockout mutants; a specific antibody should detect a band of the expected molecular weight only in wild-type samples. Second, conduct immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein. Third, perform immunolocalization studies to verify that the detection pattern matches the expected subcellular distribution of NB-ARC proteins, which typically show both cytoplasmic and nuclear localization . Fourth, test antibody specificity against recombinant AT4G27220 and closely related NLR proteins to evaluate cross-reactivity. Finally, use RNA interference or CRISPR-induced knockdowns with varying degrees of AT4G27220 suppression to establish a quantitative relationship between protein expression levels and antibody signal strength.

What expression systems are most suitable for producing recombinant AT4G27220 for antibody production and validation?

For recombinant AT4G27220 production, several expression systems offer distinct advantages. Plant-based expression systems like Nicotiana benthamiana provide the most native post-translational modifications and proper protein folding for plant proteins . The transient expression via Agrobacterium infiltration allows rapid production within 3-5 days. For higher yield production, consider using Pichia pastoris, which has successfully expressed functional plant resistance proteins with appropriate folding . For producing specific domains rather than the full-length protein, E. coli systems (particularly using solubility tags like MBP or SUMO) can be effective for the NB-ARC domain. If expressing the full-length protein proves challenging, fragmenting the protein into functional domains (NB-ARC domain separately from LRR domain) often improves solubility while maintaining native epitopes. Codon optimization for the chosen expression system and inclusion of protease inhibitors during purification are critical for success with these complex proteins.

What are the optimal protein extraction methods for preserving AT4G27220 epitopes for immunodetection?

The optimal protein extraction method for AT4G27220 must preserve protein structure while maximizing yield. Use a buffer containing 100 mM Tris (pH 7.8), 200 mM NaCl, 1 mM EDTA, 2% (v/v) β-mercaptoethanol, and 0.2% (v/v) Triton X-100, similar to protocols used for other plant NLR proteins . Add a comprehensive protease inhibitor cocktail immediately before use to prevent degradation. Perform extraction at 4°C to preserve protein integrity. For membrane-associated fractions of AT4G27220, include 1% digitonin or 0.5% NP-40 to solubilize membrane-bound proteins without excessive denaturation. Use fresh tissue whenever possible, as NLR proteins can degrade during storage. For immunoprecipitation applications, consider a gentler lysis approach using 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 10% glycerol. Filter lysates through cheesecloth followed by centrifugation at 20,000 × g for 15 minutes to remove debris while preserving protein complexes that may contain AT4G27220.

How can researchers optimize immunoblotting protocols for detecting low-abundance AT4G27220 protein?

Optimizing immunoblotting for low-abundance AT4G27220 requires several technical refinements. Start with at least 50-100 μg of total protein extract to ensure adequate target protein amounts. Use 8% SDS-PAGE gels to achieve optimal resolution for this large protein (approximately 100-150 kDa) . For protein transfer, employ a semi-dry transfer system at 200 mA for 2 hours at room temperature using 0.45 μm nitrocellulose membrane. Block membranes with a combination of 5% milk powder and 1% BSA in TBS-T overnight at 4°C to reduce background while preserving specific binding sites . Dilute primary antibodies to 1:1000-1:2000 and incubate for extended periods (overnight at 4°C) to maximize binding to scarce target protein. Utilize high-sensitivity chemiluminescent detection systems capable of detecting femtogram-range protein quantities, with exposure times of 2-5 minutes. For particularly challenging samples, consider signal amplification using biotinylated secondary antibodies followed by streptavidin-HRP complexes, which can enhance sensitivity by 5-10 fold compared to standard secondary antibodies.

What immunolocalization techniques are most effective for studying AT4G27220 distribution in plant cells?

For effective immunolocalization of AT4G27220, begin with proper sample fixation using 4% paraformaldehyde to preserve cellular architecture while maintaining epitope accessibility . For Arabidopsis, fix tissue samples for 1-2 hours at room temperature under vacuum to ensure fixative penetration. After fixation, perform careful cell permeabilization using 0.1-0.5% Triton X-100 for 30 minutes, as incomplete permeabilization is a common cause of false negatives with nuclear-localized proteins like some NLRs. Block with 3% fish skin gelatin combined with 1% BSA in PBST to reduce background fluorescence . Use primary antibody at 1:400 dilution and incubate for 12-16 hours at 4°C. For dual labeling, combine with antibodies against subcellular markers like nuclear lamina or chromatin to provide context for AT4G27220 localization. Confocal microscopy with z-stack acquisition is essential to determine the true subcellular distribution, particularly for distinguishing between nuclear and perinuclear localization. Include controls visualizing chromocenters with DAPI staining, as NLR proteins have been implicated in chromocenter organization and nuclear architecture .

How should researchers quantify and normalize AT4G27220 protein levels across different experimental conditions?

Quantification of AT4G27220 protein levels requires careful normalization strategies. For western blot analysis, use internal loading controls specific to the cellular compartment where AT4G27220 is being studied—histone H3 for nuclear fractions and actin or tubulin for cytoplasmic fractions. Avoid using housekeeping genes whose expression might change during pathogen challenge. Perform densitometric analysis using software like ImageJ with background subtraction and normalize AT4G27220 signal to the loading control signal. For accuracy, create a standard curve using recombinant AT4G27220 protein at known concentrations to establish the linear range of detection. When comparing samples across multiple blots, include a common reference sample on each blot for inter-blot normalization. For immunofluorescence quantification, measure mean fluorescence intensity within defined cellular compartments, subtracting background values from adjacent areas lacking specific signal. Report data as fold-change relative to appropriate controls rather than absolute values to account for variations in antibody affinity and detection efficiency.

What statistical approaches are most appropriate for analyzing changes in AT4G27220 protein expression during immune responses?

Statistical analysis of AT4G27220 expression changes requires approaches that account for the typically high variability in plant immune responses. For time-course experiments, apply repeated measures ANOVA followed by appropriate post-hoc tests (such as Tukey's HSD) to identify significant time points of expression change. When comparing multiple genotypes or treatments, use factorial ANOVA to assess potential interaction effects between genotype and treatment. For non-normally distributed data (common with protein quantification), apply non-parametric alternatives such as Kruskal-Wallis followed by Dunn's post-hoc test. Include at least 3-4 biological replicates (separate plants) and 2-3 technical replicates for each condition to achieve adequate statistical power. Calculate coefficient of variation to ensure methodological consistency across replicates (aim for CV < 25%). When integrating protein expression data with phenotypic outcomes (disease resistance measurements), employ correlation analyses or regression models to establish quantitative relationships between AT4G27220 levels and physiological responses. Consider using mixed linear models when experimental designs include multiple variables such as time, genotype, and treatment.

How can researchers distinguish between specific AT4G27220 signal and background in complex plant tissues?

Distinguishing specific AT4G27220 signal from background requires rigorous controls and analytical approaches. First, include knockout mutants (at4g27220) as negative controls in all experiments to establish baseline background levels . Second, perform peptide competition assays by pre-incubating the antibody with the immunizing peptide before application to samples; a specific signal should be significantly reduced. Third, use multiple antibodies targeting different regions of AT4G27220 when possible; co-localization of signals increases confidence in specificity. Fourth, when performing immunohistochemistry on tissues with high autofluorescence (like leaves), use confocal microscopy with spectral unmixing to separate antibody signal from plant autofluorescence. Fifth, apply image analysis techniques such as adaptive thresholding based on signal-to-noise ratios rather than absolute intensity values. For western blots, compare the molecular weight of detected bands with theoretical predictions and recombinant protein standards. Consider differential extraction methods to separately analyze nuclear and cytoplasmic fractions, which can help distinguish between specific localization and background contamination.

What are the common causes of false negative results when detecting AT4G27220, and how can they be addressed?

False negative results in AT4G27220 detection can arise from multiple factors. Insufficient protein extraction is a primary cause, particularly since NLR proteins can form insoluble complexes upon activation. To address this, modify extraction buffers to include a gradient of detergent concentrations (0.1% to 1% Triton X-100) to identify optimal solubilization conditions. Epitope masking through protein-protein interactions or conformational changes is another common issue, especially since NLR proteins undergo significant structural reorganization during immune activation. Try multiple antibodies targeting different epitopes or perform mild denaturation using 1-2M urea to expose hidden epitopes while preserving antibody recognition sites. Low abundance of AT4G27220, particularly in non-induced states, may require signal amplification techniques such as tyramide signal amplification for immunofluorescence or high-sensitivity ECL substrates for western blots. Sample degradation during preparation can eliminate detection; always prepare fresh samples and maintain a continuous cold chain with protease inhibitors. Finally, certain fixatives may destroy AT4G27220 epitopes; compare results using different fixation methods (paraformaldehyde, glutaraldehyde, methanol) to determine optimal epitope preservation.

How can researchers address cross-reactivity issues with AT4G27220 antibodies?

Addressing cross-reactivity of AT4G27220 antibodies requires systematic troubleshooting and validation. First, perform comprehensive sequence alignment of the immunizing peptide or protein fragment against the Arabidopsis proteome to identify potential cross-reactive proteins. Test antibody specificity against recombinant proteins from closely related NLR family members, particularly those with high sequence similarity in the targeted region. Consider immunodepletion approaches where the antibody is pre-adsorbed with recombinant proteins of potential cross-reactive targets before use in experiments. For western blots, optimize SDS-PAGE conditions to achieve maximum separation between AT4G27220 and similarly sized proteins; gradient gels (5-15%) often provide better resolution of complex samples. Increase washing stringency by using higher salt concentrations (up to 500 mM NaCl) in wash buffers to reduce non-specific binding. For particularly problematic antibodies, affinity purification against the specific immunizing peptide can dramatically improve specificity. Finally, validate all findings using genetic approaches (knockout/knockdown lines) and multiple antibodies when possible to confirm observations.

What strategies can improve detection of conformational changes in AT4G27220 during immune activation?

Detecting conformational changes in AT4G27220 during immune activation requires specialized approaches beyond standard immunodetection. Consider developing conformation-specific antibodies by immunizing with AT4G27220 in different nucleotide-bound states (ATP vs. ADP) that stabilize distinct conformations. Native PAGE rather than SDS-PAGE can preserve protein complexes and conformational states; combine with western blotting for sensitive detection. Limited proteolysis assays can reveal differential protease accessibility patterns between active and inactive conformations. Fluorescence resonance energy transfer (FRET) using antibody fragments conjugated to donor and acceptor fluorophores can detect conformational changes based on altered spatial arrangements of epitopes. For in situ analysis, proximity ligation assays can detect changes in the spatial relationship between different domains of AT4G27220 during activation. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) combined with immunoprecipitation using AT4G27220 antibodies can provide detailed information about conformational dynamics during immune activation. Develop a panel of monoclonal antibodies targeting different epitopes to create a "conformational fingerprint" that changes during activation.

How can researchers use AT4G27220 antibodies to study protein-protein interactions in plant immunity?

For studying AT4G27220 protein-protein interactions, co-immunoprecipitation (co-IP) with stringently validated antibodies provides a powerful approach. Optimize IP conditions using 0.1-0.5% digitonin or 1% NP-40 to preserve protein complexes while solubilizing membrane-associated components. Cross-linking with formaldehyde (0.5-1%) before extraction can stabilize transient interactions that might otherwise be lost during purification. Perform reciprocal co-IPs with antibodies against suspected interaction partners to confirm associations. For detecting dynamic interactions during immune responses, conduct time-course experiments after pathogen treatment, collecting samples at intervals from 0 to 24 hours post-infection. Combine co-IP with mass spectrometry for unbiased identification of novel interaction partners, using label-free quantification to determine enrichment over controls. Proximity-dependent biotin identification (BioID) using antibody-guided targeting can identify proteins in the vicinity of AT4G27220 in vivo. For spatial resolution, bimolecular fluorescence complementation (BiFC) with split fluorescent proteins can visualize interactions in specific subcellular compartments. Analyze how these interactions change in different genetic backgrounds (e.g., in adf4 mutants) to uncover regulatory mechanisms .

What approaches can reveal the role of AT4G27220 in chromatin remodeling and nuclear organization?

Investigating AT4G27220's role in chromatin remodeling requires specialized nuclear techniques. Begin with chromatin immunoprecipitation (ChIP) using AT4G27220 antibodies to identify genomic regions associated with the protein, followed by sequencing (ChIP-seq) for genome-wide binding profiles. Combine this with ATAC-seq to determine if AT4G27220 binding correlates with changes in chromatin accessibility during immune responses. Develop imaging approaches using super-resolution microscopy with AT4G27220 antibodies to visualize association with chromocenters, as NLR proteins have been implicated in chromocenter organization . Perform co-immunostaining with histone modification markers (H3K9me2, H3K27me3) to determine relationships between AT4G27220 localization and specific chromatin states. Use proximity ligation assays to detect interactions with known chromatin remodelers and modifiers. Investigate how mutations in AT4G27220 affect nuclear architecture by quantifying parameters such as chromocenter size, number, and distribution . Perform nuclear fractionation to separate euchromatin and heterochromatin, followed by immunoblotting for AT4G27220, to determine preferential association with specific chromatin types. Explore potential RNA-binding activity through RNA immunoprecipitation, as some NLRs interact with RNA components of chromatin.

How can AT4G27220 antibodies be used to investigate the relationship between plant cytoskeleton dynamics and immune signaling?

Investigating connections between AT4G27220 and cytoskeletal dynamics requires integrative approaches. Perform co-immunolocalization of AT4G27220 with actin and microtubule markers to establish spatial relationships, particularly during pathogen challenge. Use time-lapse imaging combined with immunofluorescence to track dynamic changes in this relationship during immune activation. Since mutations in actin depolymerizing factor genes (adf4) affect both cytoskeletal organization and NLR gene expression, including AT4G27220 , investigate how perturbations to actin dynamics influence AT4G27220 localization and function. Employ proximity biotinylation approaches with antibody-guided targeting to identify cytoskeletal proteins in close proximity to AT4G27220. Perform co-immunoprecipitation studies to detect direct interactions with cytoskeletal regulators, particularly focusing on actin-binding proteins implicated in immune responses. Combine these immunological approaches with live-cell imaging of cytoskeletal dynamics in plants with altered AT4G27220 expression to establish causal relationships. For mechanistic studies, use in vitro reconstitution assays with purified components to determine if AT4G27220 directly influences actin polymerization, bundling, or severing activities. Investigate potential mechanosensing functions by combining immunodetection with controlled physical perturbations to the cytoskeleton.