CRK36 Antibody

What is the molecular function of CRK36 in plant immune signaling?

CRK36 functions as a positive regulator in plant innate immunity by enhancing hypersensitive cell death, ROS burst, and disease resistance. Research has demonstrated that CRK36 directly interacts with and induces phosphorylation of BIK1 in response to flg22 treatment . CRK36 forms associations with FLS2 and BIK1 in vivo, contributing to a signaling complex that enhances pattern-triggered immunity responses, particularly stomatal defense . The protein appears to form a positive activation loop with BIK1 and NADPH oxidases that amplifies ROS production, ultimately promoting stomatal immunity against pathogens .

What structural features of CRK36 should be considered when selecting antibodies?

CRK36 contains distinctive DUF26 motifs with critical cysteine residues that are essential for its function. Studies have shown that mutations in these cysteine residues within the DUF26 motifs disrupt CRK36's ability to enhance flg22-triggered BIK1 phosphorylation . The protein possesses both extracellular domains (containing the DUF26 motifs) and an intracellular kinase domain. When selecting antibodies, researchers should consider which protein domain is most relevant to their experimental questions - antibodies targeting the extracellular domain may be more suitable for localization studies, while those recognizing the kinase domain might be better for activity studies .

How is CRK36 expression regulated during pathogen challenge?

CRK36 was originally identified through cDNA microarray analysis as a gene highly responsive to the necrotrophic fungal pathogen Alternaria brassicicola . Expression studies using promoter-GUS fusions have revealed that CRK36 is expressed in multiple tissues including hypocotyls, roots, leaves (particularly older and senescing leaves), hydathodes, and reproductive organs like sepals and septum tips . CRK36 expression increases significantly upon treatments with various pathogens including A. brassicicola and different strains of Pseudomonas syringae . Additionally, CRK36 transcription is upregulated in response to defense-related phytohormones including salicylic acid, methyl jasmonate, and ethephon (an ethylene releaser) .

What are the optimal methods for validating CRK36 antibody specificity?

To validate CRK36 antibody specificity, researchers should implement a multi-faceted approach:

• Genetic validation using different CRK36 expression lines:

  • Compare antibody reactivity between wild-type plants, crk36 knockdown mutants, and CRK36OE overexpression lines

  • Verify detection of appropriate band intensity differences proportional to expression levels

  • Include negative controls from related but distinct CRK family members

• Biochemical validation:

  • Perform peptide competition assays using the immunizing peptide

  • Test antibody reactivity under both reducing and non-reducing conditions

  • Confirm that antibody detects recombinant CRK36 protein at expected molecular weight

• Application-specific validation:

  • For Western blots: Confirm single band at the predicted molecular weight (~65 kDa)

  • For immunolocalization: Compare localization patterns between wild-type and mutant lines

  • For immunoprecipitation: Verify enrichment of CRK36 in IP vs. input samples

These validation steps are critical as the CRK family comprises multiple members with sequence similarity that could potentially cross-react with antibodies .

How can CRK36 antibodies be utilized to study protein-protein interactions in immune complexes?

CRK36 antibodies provide valuable tools for investigating its interactions with immune complex components:

• Co-immunoprecipitation approaches:

  • Use CRK36 antibodies to pull down complexes followed by immunoblotting for FLS2 and BIK1

  • Research has demonstrated that CRK36 associates with both FLS2 and BIK1 in vivo

  • Include appropriate negative controls (IgG, unrelated kinases)

  • Consider membrane solubilization conditions that preserve protein-protein interactions

• Stimulus-dependent interaction studies:

  • Monitor changes in CRK36-FLS2-BIK1 interactions following flg22 treatment

  • Compare interactions in wild-type vs. mutant backgrounds (e.g., fls2, bik1, rbohD/F)

  • Explore how pathogen challenge alters complex formation dynamics

• Localization studies:

  • Co-localization immunofluorescence with antibodies against interaction partners

  • Proximity ligation assays to visualize direct protein-protein interactions in situ

Research has shown that CRK36-mediated immune responses are dependent on both BIK1 and NADPH oxidases (RbohD/RbohF), making these proteins important targets for interaction studies .

What methodological approaches can detect CRK36 phosphorylation state changes?

Detecting CRK36 phosphorylation changes during immune responses requires specialized techniques:

• Phosphorylation-specific detection methods:

  • Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated CRK36

  • Mobility shift assays under standard SDS-PAGE conditions

  • Phospho-specific antibodies (if available)

• Kinase activity assays:

  • Immunoprecipitate CRK36 followed by in vitro kinase assays

  • Monitor changes in phosphorylation status following flg22 treatment

  • Compare kinase activity between wild-type CRK36 and mutant versions (e.g., DUF26 cysteine mutations)

• Mass spectrometry approaches:

  • Immunoprecipitate CRK36 from control and stimulated tissues

  • Identify specific phosphorylation sites by MS/MS analysis

  • Quantify changes in phosphorylation levels

Research has shown that CRK36 enhances flg22-triggered BIK1 phosphorylation, and this effect is disrupted when cysteine residues in the DUF26 motifs are mutated , making phosphorylation state analysis particularly relevant.

What are the optimal sample preparation methods for CRK36 immunodetection in plant tissues?

Effective sample preparation is crucial for successful CRK36 immunodetection:

• Protein extraction buffer optimization:

  • Use buffers containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100

  • Include protease inhibitors (PMSF, protease inhibitor cocktail)

  • Add phosphatase inhibitors (NaF, Na₃VO₄) when studying phosphorylation events

  • Consider reducing agents (DTT, β-mercaptoethanol) to maintain DUF26 domain integrity

• Tissue-specific considerations:

  • For leaf tissue: Rapid freezing in liquid nitrogen followed by grinding

  • For roots: Additional washing steps to remove soil contaminants

  • For microscopy: Fixation with 4% paraformaldehyde for cellular structure preservation

• Membrane protein extraction:

  • As CRK36 is a transmembrane protein, include adequate detergent concentrations

  • Consider membrane fractionation to enrich for plasma membrane proteins

  • Avoid harsh detergents that might disrupt protein-protein interactions

Since CRK36OE plants show enhanced ROS burst and immune responses , timing of tissue collection relative to pathogen challenge or PAMP treatment is critical for capturing relevant molecular events.

How can researchers troubleshoot inconsistent CRK36 antibody detection results across experiments?

Addressing inconsistent CRK36 detection requires systematic troubleshooting:

• Sample preparation variables:

  • Standardize plant growth conditions (light, temperature, humidity)

  • Control for developmental stage and tissue type

  • Maintain consistent extraction buffer composition

  • Ensure sample storage conditions preserve protein integrity

• Antibody-related factors:

  • Test different antibody concentrations and incubation conditions

  • Consider batch-to-batch variation in antibody preparations

  • Optimize blocking conditions to reduce background

  • Evaluate different secondary antibodies

• Protein-specific considerations:

  • CRK36 expression varies with pathogen challenge and hormone treatments

  • Expression patterns differ across tissue types and developmental stages

  • Consider protein turnover/stability factors

• Technical controls:

  • Include positive controls (CRK36OE samples) in each experiment

  • Use consistent loading controls

  • Implement quantitative normalization methods

  • Document all experimental variables systematically

Research has shown that CRK36 expression is highly responsive to pathogen infection and hormone treatments , so controlling these variables is particularly important for reproducible results.

What controls are essential when using CRK36 antibodies for immunolocalization studies?

Robust immunolocalization studies with CRK36 antibodies require comprehensive controls:

• Genetic controls:

  • Compare signal patterns between wild-type, crk36 knockdown, and CRK36OE plants

  • Include tissue from closely related CRK mutants to assess specificity

• Technical controls:

  • Secondary antibody-only controls to assess background fluorescence

  • Peptide competition/pre-absorption to confirm specific binding

  • Autofluorescence controls, particularly important in plant tissues

  • Nuclear and membrane counterstains for subcellular localization reference

• Biological controls:

  • Compare unstimulated vs. pathogen/PAMP-treated tissues

  • Include tissues known to have differential CRK36 expression (e.g., hydathodes, senescing leaves)

  • Consider developmental stage effects on localization patterns

Research has demonstrated that CRK36 interacts with plasma membrane-localized proteins like FLS2 and BIK1 , making membrane localization studies particularly informative.

How should researchers interpret differences in CRK36 detection between various mutant backgrounds?

Interpreting CRK36 detection across different genetic backgrounds requires careful analysis:

• Expression level differences:

  • crk36 knockdown lines show 30-50% reduction in CRK36 transcript levels compared to wild-type

  • CRK36OE lines exhibit significantly increased expression

  • Quantify relative protein abundance using densitometry of Western blots

  • Consider how genetic background might affect post-translational modifications

• Phenotypic correlation analysis:

  • CRK36OE plants show enhanced cell death, ROS production, and disease resistance

  • crk36 plants display weakened immune responses

  • Correlate protein detection levels with observed immunity phenotypes

• Pathway component analysis:

  • Examine CRK36 detection in backgrounds with mutations in interacting partners (fls2, bik1, rbohD/F)

  • Research has shown that disruption of BIK1 and RbohD/RbohF impairs CRK36-mediated stomatal defense

  • Consider feedback regulation effects on CRK36 expression/stability

• Statistical approaches:

  • Perform multiple biological replicates (n≥3)

  • Apply appropriate statistical tests for comparing detection levels

  • Consider variance components from biological and technical sources

These interpretations should be integrated with functional data on immune responses to provide comprehensive understanding.

How can researchers reconcile potential discrepancies between CRK36 protein levels and transcript expression data?

Addressing discrepancies between protein and transcript levels requires careful consideration of multiple regulatory layers:

• Post-transcriptional regulation:

  • CRK36 may be subject to regulation by miRNAs or RNA-binding proteins

  • Analyze transcript stability using actinomycin D chase experiments

  • Consider alternative splicing possibilities

• Translational and post-translational regulation:

  • Examine protein half-life using cycloheximide chase experiments

  • Investigate ubiquitination status and proteasomal degradation

  • Study potential feedback regulation mechanisms

• Technical considerations:

  • Different sensitivity thresholds between RT-qPCR and immunodetection

  • Temporal delay between transcriptional changes and protein accumulation

  • Extraction efficiency differences for RNA versus protein

• Integrated analysis approaches:

  • Time course studies following pathogen challenge or PAMP treatment

  • Correlation analysis across multiple biological conditions

  • Consideration of tissue/cell-specific regulation mechanisms

Research has shown that CRK36 expression responds rapidly to pathogen challenge , so temporal dynamics are particularly important to consider when comparing transcript and protein data.

What statistical approaches are most appropriate for analyzing variability in CRK36 immunodetection across biological replicates?

Rigorous statistical analysis of CRK36 immunodetection requires appropriate methods:

• Descriptive statistics:

  • Calculate means, standard deviations, and coefficients of variation

  • Assess normality of data distribution (Shapiro-Wilk test)

  • Identify potential outliers using box plots or Dixon's Q test

• Inferential statistics:

  • For comparing multiple genotypes/treatments: ANOVA with appropriate post-hoc tests

  • For comparing two conditions: t-test or non-parametric alternatives

  • Include correction for multiple comparisons when necessary

• Variance components analysis:

  • Distinguish between biological and technical variation sources

  • Use mixed-effects models to account for hierarchical data structure

  • Consider nested experimental designs (plants within treatment groups)

• Normalization strategies:

  • Normalization to housekeeping proteins (actin, tubulin)

  • Consider total protein normalization methods (Ponceau S, SYPRO Ruby)

  • Evaluate reference gene stability across experimental conditions

Since CRK36 expression is highly responsive to stimuli , researchers should account for treatment-dependent variability when designing statistical approaches.

How can CRK36 antibodies be utilized to investigate crosstalk between biotic and abiotic stress responses?

CRK36 antibodies enable investigation of stress response crosstalk through several approaches:

• Comparative protein analysis:

  • Monitor CRK36 protein levels following combined biotic and abiotic stresses

  • Compare post-translational modifications (phosphorylation, glycosylation)

  • Examine protein complex formation under different stress conditions

• Subcellular localization studies:

  • Track CRK36 localization changes during combined stresses

  • Investigate co-localization with stress-specific markers

  • Examine membrane microdomain association during different stresses

• Interaction partner identification:

  • Immunoprecipitate CRK36 following different stresses

  • Identify unique or shared interaction partners by mass spectrometry

  • Verify interactions with co-immunoprecipitation or BiFC

Research has shown that CRK36 responds to pathogen infection and also to hormones like SA, MeJA, and ethylene , suggesting potential roles in multiple stress response pathways.

What methodological considerations are important when using CRK36 antibodies to study specific mutations in the DUF26 motifs?

Studying DUF26 motif mutations requires specialized methodological approaches:

• Antibody epitope considerations:

  • Determine if antibody epitopes overlap with mutated regions

  • Use multiple antibodies targeting different protein regions

  • Compare detection efficiency between wild-type and mutant proteins

• Structural analysis implications:

  • Research has shown that Cys mutations in DUF26 motifs disrupt CRK36 function

  • Consider how these mutations affect protein folding and stability

  • Examine disulfide bond formation under reducing/non-reducing conditions

• Functional correlation studies:

  • Compare immunodetection results with functional assays (ROS production, pathogen resistance)

  • Examine interaction with BIK1 and impact on its phosphorylation

  • Investigate membrane localization changes due to DUF26 mutations

• Experimental design considerations:

  • Include appropriate controls (wild-type CRK36, other DUF26 mutants)

  • Consider transient expression systems for comparing multiple mutant variants

  • Use complementary approaches (fluorescent tagging and antibody detection)

These approaches can provide insights into the structural basis of CRK36 function in immune signaling.

How can CRK36 antibodies contribute to understanding the dynamics of PRR complex formation during immune responses?

CRK36 antibodies provide valuable tools for studying pattern recognition receptor (PRR) complex dynamics:

• Temporal analysis approaches:

  • Immunoprecipitate CRK36 at different time points following PAMP treatment

  • Monitor association with FLS2 and BIK1 over time

  • Correlate complex formation with downstream signaling events

• Compositional analysis:

  • Research has demonstrated that CRK36 associates with both FLS2 and BIK1 in vivo

  • Use sequential immunoprecipitation to identify subcomplexes

  • Employ quantitative proteomics to determine stoichiometry changes

• Functional analysis:

  • Compare complex formation in wild-type vs. immune-compromised backgrounds

  • Correlate complex dynamics with ROS production and stomatal immunity

  • Investigate how complex composition affects BIK1 phosphorylation

• Spatial organization studies:

  • Examine membrane microdomain localization of complexes

  • Study co-localization patterns before and after stimulation

  • Investigate potential endocytosis following activation

These approaches can reveal how CRK36 contributes to the dynamic assembly and function of immune receptor complexes at the plant cell surface.