At5g56590 Antibody

What is AT5G16590 and why is it significant in plant research?

AT5G16590 encodes a probable inactive receptor kinase in Arabidopsis thaliana that contains leucine-rich repeat (LRR) domains. This protein belongs to the family of LRR receptor-like proteins that play crucial roles in plant immune responses, particularly in pathogen recognition and signal transduction. Antibodies targeting this protein are valuable tools for studying plant-pathogen interactions, especially in the context of receptor-mediated immunity . The significance of this protein lies in its potential role in perceiving apoplastic effectors from pathogens such as Hyaloperonospora arabidopsidis (Hpa), the causative agent of downy mildew in Arabidopsis .

Which plant species can AT5G16590 antibody detect?

The AT5G16590 antibody demonstrates cross-reactivity with proteins from multiple plant species. According to specificity data, the antibody labeled PHY0947A recognizes homologous proteins in Arabidopsis thaliana, Glycine max (soybean), Brassica rapa, Brassica napus, Medicago truncatula, Vitis vinifera (grapevine), Populus trichocarpa, Gossypium raimondii, Nicotiana tabacum (tobacco), Cucumis sativus (cucumber), Spinacia oleracea (spinach), Solanum tuberosum (potato), and Solanum lycopersicum (tomato) . The broad cross-reactivity makes this antibody valuable for comparative studies across different plant species.

How should AT5G16590 antibody be stored and handled in a laboratory setting?

Proper storage and handling of the AT5G16590 antibody is critical for maintaining its activity and specificity. The lyophilized antibody should be stored in a manual defrost freezer to prevent degradation. Upon receipt, it should be immediately stored at the recommended temperature. To maximize shelf life, avoid repeated freeze-thaw cycles which can damage the antibody structure. The product is typically shipped at 4°C but requires proper storage upon arrival . For reconstitution, follow the manufacturer's specific guidelines regarding buffer composition and concentration to ensure optimal antibody performance in experimental applications.

What are the optimal immunodetection methods for AT5G16590 in plant tissues?

For effective immunodetection of AT5G16590 in plant tissues, researchers should consider both the protein's subcellular localization and abundance. As a membrane-associated receptor kinase, standard protein extraction protocols may require optimization with specialized buffers containing appropriate detergents to solubilize membrane proteins. For immunoblotting, a recommended approach involves:

• Extracting total proteins from plant tissues using a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and protease inhibitor cocktail

• Separating proteins via SDS-PAGE with 8-10% acrylamide gels (optimal for resolving proteins in the 70-100 kDa range)

• Transferring to PVDF membranes at 100V for 60 minutes in cold transfer buffer

• Blocking with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubating with primary AT5G16590 antibody (1:1000 dilution) overnight at 4°C

• Washing and detecting with appropriate secondary antibody and visualization system

For immunolocalization studies, tissue fixation protocols should be optimized to preserve protein epitopes while allowing antibody penetration .

How can AT5G16590 antibody be used to investigate plant-pathogen interactions?

AT5G16590 antibody can be instrumental in studying plant-pathogen interactions, particularly in the context of receptor-mediated immunity. Methodological approaches include:

• Co-immunoprecipitation assays: To identify potential interacting partners of AT5G16590 during pathogen infection, the antibody can be used to pull down the receptor complex from infected plant tissues. This approach can help identify both plant proteins in the receptor complex and potential pathogen effectors that interact with the receptor.

• Protein localization studies: Immunofluorescence microscopy using the AT5G16590 antibody can reveal changes in receptor localization during pathogen challenge, providing insights into receptor dynamics during immune responses.

• Protein accumulation analysis: Western blotting with the AT5G16590 antibody can quantify changes in receptor protein levels in response to pathogen infection or treatment with pathogen-associated molecular patterns (PAMPs).

• Receptor activation studies: The antibody can be used to immunoprecipitate the receptor for subsequent analysis of post-translational modifications, such as phosphorylation, which may indicate receptor activation during immune responses .

What controls should be included when performing immunoblotting with AT5G16590 antibody?

When conducting immunoblotting experiments with AT5G16590 antibody, the following controls are essential for data validation:

The inclusion of these controls is particularly important given the reported sequence homology (93% for PHY0947A and 87% for PHY3606S) between AT5G16590 and KIN7 (AT3G02880) . This comprehensive control setup will help distinguish specific signals from potential cross-reactivity or background.

How can AT5G16590 antibody be utilized in receptor complex immunoprecipitation studies?

For advanced receptor complex immunoprecipitation studies using AT5G16590 antibody, a methodological approach would involve:

• Sample preparation: Cross-link proteins in intact plant tissues or cultured cells using membrane-permeable crosslinkers (e.g., DSP or formaldehyde) to stabilize transient protein-protein interactions within receptor complexes.

• Membrane protein extraction: Utilize specialized buffers containing 1% digitonin or 0.5% n-dodecyl-β-D-maltoside, which effectively solubilize membrane proteins while preserving protein-protein interactions.

• Immunoprecipitation: Conjugate AT5G16590 antibody to protein A/G magnetic beads or appropriate affinity matrix. Pre-clear lysates to reduce non-specific binding, then incubate with antibody-conjugated beads overnight at 4°C with gentle rotation.

• Washing and elution: Perform stringent washing steps with decreasing detergent concentrations to remove non-specific interactions while preserving specific complex components. Elute proteins using low pH buffer or SDS-containing buffer.

• Complex analysis: Analyze precipitated complexes using mass spectrometry to identify interacting partners. Perform reciprocal co-immunoprecipitation with antibodies against identified partners to confirm interactions.

This approach can be particularly valuable for investigating how AT5G16590 potentially functions in pathogen recognition, especially in the context of apoplastic effector perception during infection with pathogens like Hyaloperonospora arabidopsidis .

What considerations are important when using AT5G16590 antibody for comparative studies across different plant species?

When utilizing AT5G16590 antibody for comparative studies across different plant species, several critical considerations must be addressed:

• Sequence homology assessment: While the antibody cross-reacts with homologous proteins in multiple plant species, researchers should perform bioinformatic analyses to determine the degree of conservation in the epitope region across target species. Higher sequence divergence may result in reduced antibody affinity.

• Validation in each species: Before conducting comparative experiments, validate antibody specificity in each species through Western blotting using both wild-type and, where available, mutant/knockout lines. This confirms that observed signals represent the intended target protein.

• Optimized extraction protocols: Different plant species contain varying levels of compounds that may interfere with antibody binding (e.g., phenolics, polysaccharides). Extraction protocols should be optimized for each species to minimize interference.

• Protein loading normalization: When comparing protein levels across species, ensure accurate normalization using appropriate loading controls verified to be consistent across the species being studied.

• Epitope accessibility considerations: Variations in post-translational modifications or protein complex formation across species may affect epitope accessibility. Consider using multiple antibody-based techniques (Western blot, immunoprecipitation, immunolocalization) to obtain comprehensive data .

How can potential cross-reactivity with KIN7 (AT3G02880) be addressed in AT5G16590 antibody experiments?

The reported sequence homology between AT5G16590 and KIN7 (AT3G02880) presents a potential challenge for experimental specificity. To address this cross-reactivity:

• Pre-absorption protocol: Incubate the AT5G16590 antibody with recombinant KIN7 protein prior to experimental use. This pre-absorption step can sequester antibodies that bind to shared epitopes, enhancing specificity for unique AT5G16590 epitopes.

• Genetic controls: Include protein samples from both AT5G16590 and KIN7 knockout mutants to establish signal contribution from each protein.

• Epitope mapping: Determine the exact epitope recognized by the antibody through peptide array analysis or hydrogen-deuterium exchange mass spectrometry. This information can guide the design of blocking peptides for enhanced specificity.

• Differential expression analysis: In some experimental systems, the differential expression patterns of AT5G16590 and KIN7 can be leveraged to interpret results correctly. Document tissue- or condition-specific expression of both proteins.

• Western blot band discrimination: Given potential molecular weight differences between the two proteins, careful calibration of SDS-PAGE conditions may allow for separation and distinct identification of each protein .

What are common challenges in immunodetection of AT5G16590 and how can they be addressed?

Researchers commonly encounter several challenges when working with AT5G16590 antibody:

• Low signal intensity: This may result from low endogenous expression of AT5G16590. Address by:

  • Enriching membrane fractions before immunoblotting

  • Using high-sensitivity detection systems (e.g., enhanced chemiluminescence)

  • Increasing antibody concentration or incubation time

  • Applying signal amplification systems

• High background: May result from non-specific binding. Mitigate by:

  • Increasing blocking agent concentration (5-10% BSA or milk)

  • Adding 0.1% Tween-20 to washing buffers

  • Diluting primary antibody in blocking buffer with 0.05% Tween-20

  • Pre-absorbing antibody with plant tissue extract from knockout mutants

• Multiple bands: Could indicate splice variants, post-translational modifications, or cross-reactivity. Differentiate by:

  • Comparing band patterns with predicted molecular weights

  • Analyzing band patterns in knockout/knockdown plants

  • Using phosphatase treatment to identify phosphorylated forms

  • Employing peptide competition assays to confirm specificity

How should researchers interpret variations in AT5G16590 detection during pathogen infection studies?

When interpreting variations in AT5G16590 detection during pathogen infection studies, researchers should consider multiple factors that might influence results:

• Temporal dynamics: AT5G16590 expression and protein levels may fluctuate throughout the infection cycle. Establish a detailed time course analysis, as some pathogen effector proteins may be deployed at specific infection stages to modulate host immune responses .

• Subcellular relocalization: Receptor kinases can undergo subcellular relocalization upon activation. Changes in detection might reflect protein redistribution rather than abundance changes. Complement immunoblotting with subcellular fractionation and immunolocalization studies.

• Post-translational modifications: Infection may trigger phosphorylation, ubiquitination, or other modifications of AT5G16590, potentially altering antibody recognition. Employ phospho-specific antibodies or analyze mobility shifts indicative of modifications.

• Proteolytic processing: Some receptors undergo cleavage upon activation or as part of pathogen suppression strategies. Unexpected band patterns may represent biologically relevant receptor fragments rather than degradation artifacts.

• Pathogen interference: Consider whether pathogen effectors directly target AT5G16590 for degradation or modification as an immune evasion strategy, particularly in compatible interactions .

What analytical methods can complement AT5G16590 antibody-based detection for comprehensive protein function studies?

For comprehensive studies of AT5G16590 function, antibody-based detection should be complemented with multiple analytical approaches:

• Transcriptional analysis: Quantitative RT-PCR and RNA-seq can correlate protein detection with transcript levels, helping distinguish between transcriptional and post-transcriptional regulation of AT5G16590.

• Genetic approaches: CRISPR/Cas9-mediated gene editing, T-DNA insertion lines, or RNAi-based silencing provide essential loss-of-function contexts for interpreting antibody-based observations. Complementation with fluorescently-tagged AT5G16590 can validate antibody specificity and provide additional localization data .

• Protein-protein interaction studies: Yeast two-hybrid, split-GFP, or FRET-based approaches can identify interaction partners, complementing co-immunoprecipitation studies with the AT5G16590 antibody.

• Structural biology: Cryo-EM or X-ray crystallography of immunopurified AT5G16590 can provide structural insights into receptor function and ligand binding properties.

• Mass spectrometry-based proteomics: Techniques such as parallel reaction monitoring (PRM) or selected reaction monitoring (SRM) can provide absolute quantification of AT5G16590 protein, while phosphoproteomics can map activation-related phosphorylation sites .

How might AT5G16590 antibody contribute to understanding apoplastic effector recognition in plants?

AT5G16590 antibody represents a valuable tool for advancing our understanding of apoplastic effector recognition in plant immunity research through several approaches:

• Effector-receptor identification: The antibody can facilitate screening for direct interactions between AT5G16590 and candidate apoplastic effectors from pathogens like Hyaloperonospora arabidopsidis. Co-immunoprecipitation followed by mass spectrometry could identify novel effector-receptor pairs, contributing to our understanding of how plants perceive pathogen molecules in the apoplast .

• Receptor complex dynamics: Immune receptors rarely function alone. The AT5G16590 antibody can help characterize dynamic changes in receptor complex composition before and after exposure to apoplastic effectors, revealing mechanisms of signal transduction.

• Spatial distribution analysis: Immunolocalization using the AT5G16590 antibody can map the distribution of this receptor in relation to infection sites and pathogen structures, potentially identifying specialized membrane domains involved in pathogen sensing.

• Structural immunology: The antibody could be used to purify native AT5G16590 protein for structural studies, providing insights into recognition mechanisms and potentially guiding the development of synthetic molecules that can trigger immunity .

What are potential applications of AT5G16590 antibody in studies of receptor-mediated immunity across plant families?

The broad cross-reactivity of AT5G16590 antibody across multiple plant species opens exciting possibilities for comparative studies of receptor-mediated immunity:

• Evolutionary conservation analysis: By examining AT5G16590 homologs across diverse plant families, researchers can investigate the evolutionary conservation of this receptor and its signaling pathways, potentially identifying core components of plant immunity that have been preserved across millions of years of evolution.

• Crop immunity enhancement: Knowledge gained from comparative studies can inform strategies for enhancing disease resistance in crops. The antibody can help validate whether orthologous receptors in crop species function similarly to AT5G16590 in Arabidopsis.

• Host-range determinant studies: For pathogens with restricted host ranges, AT5G16590 antibody can help determine whether differences in receptor structure, abundance, or localization correlate with susceptibility or resistance across plant species.

• Receptor transfer validation: In transgenic approaches where AT5G16590 or its orthologs are transferred between plant species, the antibody can confirm proper expression, localization, and function of the transferred receptor .

How can AT5G16590 antibody be integrated with emerging technologies for plant immune receptor research?

Integration of AT5G16590 antibody with cutting-edge technologies can significantly advance plant immune receptor research:

• Single-cell proteomics: Coupling the AT5G16590 antibody with emerging single-cell proteomics techniques could reveal cell-type-specific variations in receptor abundance and activation status, particularly at infection sites where cellular responses may be heterogeneous.

• Super-resolution microscopy: Combining fluorescently-labeled AT5G16590 antibody with techniques like STORM or PALM microscopy can visualize nanoscale receptor clustering and organization in the plasma membrane during immune responses.

• Proximity labeling: Using the antibody to validate BioID or APEX2-based proximity labeling approaches can map the dynamic interactome of AT5G16590 in living cells, capturing even transient interactions during immune signaling.

• Organ-on-chip platforms: AT5G16590 antibody-based detection can be integrated into microfluidic "plant-on-chip" platforms to monitor receptor dynamics during precisely controlled pathogen challenges.

• CRISPR-based receptor engineering: The antibody provides an essential validation tool for CRISPR-engineered variants of AT5G16590, allowing researchers to confirm that edited receptors are properly expressed and localized before phenotypic analysis .