CPK28 Antibody

What is CPK28 and why is it significant in plant research?

CPK28 is a calcium-dependent protein kinase that functions as an important sensor-responder protein in Arabidopsis. It binds Ca²⁺ to sense intracellular calcium levels and responds by phosphorylating target proteins . CPK28 plays dual critical roles in plant biology: it negatively regulates immune responses against bacterial pathogens while positively regulating plant growth and development . This dual functionality makes CPK28 a crucial component in understanding how plants balance growth and defense mechanisms. Specifically, CPK28 regulates immune homeostasis by controlling the stability of Botrytis-Induced Kinase 1 (BIK1), a key component of plant pattern-triggered immunity . The significance of CPK28 extends to its potential applications in agricultural biotechnology, as understanding its regulation could enable breeding of pathogen-resistant crops without compromising yield .

How specific are commercially available CPK28 antibodies?

Based on experimental evidence, properly validated CPK28 antibodies demonstrate high specificity when tested against wild-type and mutant plant samples. In particular, studies have generated antibodies that specifically detect CPK28 in Arabidopsis Col-0 plants but not in cpk28 mutants . These antibodies specifically recognize recombinant CPK28 proteins but do not cross-react with other tested CPKs, including closely related proteins such as CPK8, CPK6, CPK1, CPK3, and even CPK16, which is closely related to CPK28 . Additionally, these validated antibodies do not recognize the truncated CPK28-RI isoform that exhibits impaired kinase activity . This high specificity makes properly validated CPK28 antibodies reliable tools for studying CPK28 functions in both basic and complex experimental setups.

What are the primary applications of CPK28 antibody in plant immunity research?

CPK28 antibodies serve several critical functions in plant immunity research. They are particularly valuable for:

• Protein detection and quantification: CPK28 antibodies enable immunoblotting assays to measure protein accumulation in response to treatments such as proteasome inhibitors (MG132) or protein synthesis inhibitors (cycloheximide) .

• Immunoprecipitation studies: CPK28 antibodies facilitate co-immunoprecipitation assays to identify protein-protein interactions, such as those between CPK28 and ubiquitin ligases like ATL31 and ATL6 , or other interaction partners like PSKR1 and GS2 .

• Phosphorylation state analysis: Using phospho-specific antibodies (such as α-pSer318) allows researchers to monitor the phosphorylation status of CPK28, which is critical for its function in immune signaling .

• Subcellular localization studies: When combined with immunofluorescence techniques, CPK28 antibodies help determine the protein's localization at the plasma membrane and potential relocalization during immune responses .

These applications collectively provide insights into how CPK28 regulates the balance between growth and immunity in plants, making CPK28 antibodies indispensable tools in understanding plant defense mechanisms.

What are the optimal conditions for using CPK28 antibodies in immunoprecipitation assays?

When using CPK28 antibodies for immunoprecipitation (IP) assays, several methodological considerations should be implemented for optimal results. First, researchers should consider using epitope-tagged CPK28 (such as CPK28-HA) when performing transient expression experiments in Arabidopsis protoplasts . This approach has been successfully employed to examine the ubiquitination of CPK28 in vivo, similar to methods used for BIK1 and FLS2 .

For co-immunoprecipitation studies examining CPK28 interactions with other proteins (such as ATL31/6, PSKR1, or GS2), it's advisable to use mutant versions of potential E3 ligases (e.g., ATL31 C143H145A) to prevent target degradation . This strategy enhances the stability of the interaction and improves detection. Furthermore, when investigating stimulus-dependent interactions, researchers should treat samples with appropriate elicitors (such as flg22 or PSK) before immunoprecipitation to capture dynamic changes in protein-protein associations .

Protocol optimizations should include:

• Using freshly prepared plant material or protoplasts

• Including protease and phosphatase inhibitors in extraction buffers

• Performing immunoprecipitation at 4°C to minimize protein degradation

• Using appropriate controls, including isotype controls and no-antibody controls

• Validating results with reciprocal co-immunoprecipitation using antibodies against interaction partners

These methodological considerations ensure robust and reproducible results when using CPK28 antibodies for immunoprecipitation studies.

How can researchers validate the specificity of CPK28 antibodies in their experimental systems?

Validating CPK28 antibody specificity is crucial for generating reliable experimental data. Researchers should implement a multi-faceted validation approach:

First, perform western blot analysis using wild-type Arabidopsis (Col-0) samples alongside cpk28 mutant samples as negative controls . A specific antibody will detect the expected ~58 kDa band in wild-type samples but not in the mutant. Second, test cross-reactivity against recombinant proteins of related CPKs (such as CPK8, CPK6, CPK1, CPK3, and CPK16) to ensure the antibody doesn't recognize closely related family members .

Additional validation steps should include:

• Testing recognition of different CPK28 isoforms, including the truncated CPK28-RI that exhibits impaired kinase activity

• Conducting peptide competition assays, where pre-incubation of the antibody with the immunizing peptide should abolish specific signals

• Employing transgenic plants expressing tagged CPK28 (e.g., CPK28-YFP) to confirm co-localization of antibody signals with the tagged protein

• Performing immunoprecipitation followed by mass spectrometry to confirm that the antibody captures the intended target

For phospho-specific antibodies (like those targeting phosphorylated Ser318), additional validation should include testing against non-phosphorylatable mutants (CPK28S318A) and phosphomimetic variants . Researchers should also include appropriate phosphatase treatments to confirm phospho-specificity.

By implementing these rigorous validation steps, researchers can ensure that their CPK28 antibodies are highly specific and suitable for their experimental systems.

What are the recommended fixation and immunostaining protocols for CPK28 localization studies?

For successful immunolocalization studies of CPK28, researchers should consider the following optimized protocol based on published methodologies:

Sample preparation begins with appropriate fixation, typically using 4% paraformaldehyde in PBS pH 7.4 for 20-30 minutes at room temperature for Arabidopsis seedlings or leaf tissues. This preserves protein localization while maintaining tissue integrity. After fixation, samples should be thoroughly washed with PBS to remove excess fixative.

Cell permeabilization is a critical step and can be achieved using 0.1-0.3% Triton X-100 in PBS for 15-20 minutes, allowing antibody access to intracellular CPK28. Following permeabilization, samples should be blocked with 3-5% BSA in PBS for 1-2 hours to minimize non-specific binding.

For primary antibody incubation, validated anti-CPK28 antibodies should be diluted (typically 1:100 to 1:500, depending on antibody quality) in blocking solution and incubated overnight at 4°C. After thorough washing with PBS containing 0.1% Tween-20, fluorescently-labeled secondary antibodies (matching the host species of the primary antibody) should be applied at appropriate dilutions (typically 1:200 to 1:1000) for 1-2 hours at room temperature.

When analyzing samples, confocal microscopy is recommended for optimal resolution, particularly since CPK28 localizes primarily to the plasma membrane . Researchers should include appropriate controls in every experiment, including samples from cpk28 mutants (negative control) and plants expressing fluorescently-tagged CPK28 (positive control) .

For co-localization studies with interaction partners like PSKR1 or BIK1, dual immunostaining or combining immunostaining with fluorescently-tagged proteins can provide valuable insights into the spatial organization of these regulatory complexes .

How can phospho-specific CPK28 antibodies be used to study its activation mechanisms?

Phospho-specific antibodies targeting key regulatory sites like Ser318 on CPK28 provide powerful tools for studying its activation mechanisms. These antibodies can be employed in multiple advanced research applications to elucidate how phosphorylation regulates CPK28 function in immunity and growth.

Researchers can use phospho-specific antibodies (such as α-pSer318) in western blot analysis to monitor the phosphorylation status of CPK28 under different conditions . This approach is particularly valuable when investigating how different stimuli (such as pathogen-associated molecular patterns or hormones) affect CPK28 activation. By comparing phosphorylation levels before and after treatments, researchers can identify signaling pathways that regulate CPK28 activity.

Time-course experiments combining phospho-specific antibodies with standard CPK28 antibodies enable quantification of the phosphorylation-to-total protein ratio, providing insights into the dynamics of CPK28 activation. Such experiments have revealed that BIK1 transphosphorylates CPK28 at Ser318, which is essential for its function in immune signaling but dispensable for its role in stem elongation .

Phospho-specific antibodies can also be applied in immunoprecipitation assays to isolate and identify proteins that specifically interact with the phosphorylated form of CPK28, potentially uncovering phosphorylation-dependent interactomes. When combined with in vitro kinase assays, these antibodies help validate whether mutations that mimic or prevent phosphorylation (phosphomimetic or phospho-null mutations) correctly reproduce the effects of natural phosphorylation .

For researchers investigating structure-function relationships, phospho-specific antibodies provide a means to correlate structural changes with phosphorylation status, as phosphorylation at Ser318 is known to alter CPK28 conformation and enhance its sensitivity to calcium at physiological concentrations .

What approaches can resolve contradictory results when using CPK28 antibodies across different experimental systems?

When researchers encounter contradictory results using CPK28 antibodies across different experimental systems, a systematic troubleshooting approach is necessary. First, evaluate antibody specificity across the different systems by performing western blots with appropriate positive and negative controls, including recombinant CPK28 protein and samples from cpk28 mutant plants . Different antibody lots may vary in specificity and sensitivity, so maintaining detailed records of which lots produced reliable results is crucial.

Consider the potential expression of alternative CPK28 isoforms in different tissues or under different conditions. For instance, the truncated CPK28-RI isoform with impaired kinase activity is induced after treatment with flg22 or AtPeps, and standard CPK28 antibodies might not recognize this isoform . Using antibodies that target different epitopes of CPK28 can help resolve whether discrepancies arise from the presence of alternative isoforms.

Post-translational modifications significantly affect CPK28 detection. CPK28 undergoes both phosphorylation (particularly at Ser318) and ubiquitination at multiple lysine residues . These modifications may mask epitopes or alter antibody recognition. To address this, researchers should:

• Compare results from phosphorylation-site specific antibodies with total CPK28 antibodies

• Include controls with phosphatase treatment to remove phosphorylation

• Consider the impact of proteasome inhibitors (like MG132) on CPK28 stability and detection

Experimental conditions, particularly calcium concentration, significantly impact CPK28 conformation and function. Phosphorylation at Ser318 primes CPK28 to sense calcium at physiological concentrations . Therefore, inconsistencies might arise from variations in calcium levels across experimental systems. Standardizing buffer conditions and calcium concentrations can help resolve such discrepancies.

When contradictions persist despite these approaches, researchers should consider employing complementary techniques such as mass spectrometry to unambiguously identify CPK28 and its modifications in their experimental systems.

How can CPK28 antibodies be optimized for detecting stimulus-dependent changes in protein-protein interactions?

Optimizing CPK28 antibodies for detecting stimulus-dependent protein-protein interactions requires specialized approaches that capture transient and dynamic associations. Evidence from the literature shows that interactions involving CPK28, such as those with ATL31/6 and PSKR1, are enhanced following stimulus treatment (flg22 or PSK, respectively) . To effectively capture these dynamic interactions, researchers should implement several methodological refinements.

First, timing is critical. Researchers should conduct time-course experiments to identify the optimal time points for capturing stimulus-enhanced interactions. For example, the association between CPK28 and ATL31/6 is enhanced after flg22 treatment, while the CPK28-PSKR1 interaction increases following PSK application . By collecting samples at multiple time points after stimulus application, researchers can determine when the interaction reaches its peak.

Co-immunoprecipitation protocols should be optimized for preserving weak or transient interactions. This includes:

• Using chemical crosslinking agents (like formaldehyde or DSP) to stabilize protein complexes before cell lysis

• Including phosphatase inhibitors to maintain phosphorylation-dependent interactions

• Utilizing gentle detergents (such as digitonin or CHAPS) instead of stronger ones like Triton X-100

• Performing the immunoprecipitation at physiological salt concentrations to preserve weaker interactions

For reliable detection of stimulus-dependent changes, quantitative approaches are essential. Researchers should:

• Use appropriate loading controls to normalize immunoprecipitation efficiency

• Employ quantitative western blotting with infrared or chemiluminescent detection

• Include multiple biological and technical replicates to ensure statistical robustness

• Consider bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) as complementary approaches for monitoring interactions in live cells

To minimize artifacts, researchers should use functional variants of interaction partners that reduce confounding effects. For instance, when studying CPK28 interaction with E3 ligases like ATL31, using the catalytically inactive variant ATL31 C143H145A prevents target degradation and enhances detection of the interaction .

How should researchers interpret apparent discrepancies between CPK28 protein levels and phenotypic outcomes?

Interpreting discrepancies between CPK28 protein levels and phenotypic outcomes requires careful consideration of multiple factors affecting CPK28 function beyond mere protein abundance. First, researchers must recognize that CPK28 undergoes complex post-translational regulation. CPK28 is subject to phosphorylation at sites like Ser318, which is critical for its function in immune signaling but dispensable for its role in growth regulation . Consequently, plants with similar CPK28 protein levels but different phosphorylation profiles may exhibit divergent immune phenotypes despite comparable growth characteristics.

Additionally, CPK28 undergoes ubiquitination at multiple lysine residues and subsequent proteasomal degradation, processes that are enhanced by flagellin treatment . This dynamic regulation means that steady-state protein levels may not reflect the actual functional pool of CPK28. Researchers should therefore combine measurements of total CPK28 with analyses of its ubiquitination status and turnover rate using cycloheximide chase assays .

The expression of alternative CPK28 isoforms further complicates interpretation. The truncated CPK28-RI isoform with impaired kinase activity is induced after elicitor treatments and plays a regulatory role in dynamically amplifying immune signaling . Standard antibodies may not distinguish between functional and truncated isoforms, leading to discrepancies between detected protein levels and observed phenotypes.

CPK28 functions within complex protein networks, interacting with partners like BIK1, PUB25/26, ATL31/6, PSKR1, and GS2 . The availability and abundance of these interaction partners can significantly influence CPK28 function independently of its own abundance. Comprehensive analysis should therefore include assessment of key interaction partners alongside CPK28 protein levels.

When interpreting data, researchers should implement multivariate statistical approaches that incorporate measurements of CPK28 protein levels, phosphorylation status, ubiquitination, interaction partner abundance, and calcium levels to develop more accurate predictive models of phenotypic outcomes.

What statistical methods are most appropriate for analyzing CPK28 antibody-based quantitative data?

When analyzing quantitative data generated using CPK28 antibodies, researchers should employ appropriate statistical methods that address the specific characteristics of immunoblotting and immunoprecipitation data. For western blot quantification of CPK28 protein levels, normalization is essential. Researchers should normalize band intensities to appropriate loading controls (such as actin or tubulin) and consider using specialized software like ImageJ or dedicated western blot quantification platforms that account for non-linear signal response .

For time-course experiments examining CPK28 stability or phosphorylation dynamics, researchers should employ regression analysis to determine degradation rates or phosphorylation kinetics. Half-life calculations for CPK28 can be derived from cycloheximide chase assays, where protein levels are measured at various time points after inhibiting protein synthesis .

When comparing CPK28 protein levels or phosphorylation states across different genotypes or treatments, appropriate parametric tests (t-test, ANOVA) or non-parametric alternatives (Mann-Whitney U, Kruskal-Wallis) should be selected based on data distribution. Post-hoc tests (such as Tukey's HSD or Dunnett's test) should be employed for multiple comparisons to control for family-wise error rates.

For co-immunoprecipitation experiments investigating stimulus-dependent interactions (such as CPK28 with ATL31/6 after flg22 treatment or CPK28 with PSKR1 after PSK application), paired statistical tests comparing before and after stimulus conditions within the same experimental setup are most appropriate .

Researchers should also consider implementing more sophisticated statistical approaches:

• Correlation analysis to examine relationships between CPK28 levels, phosphorylation states, and phenotypic outcomes

• Principal component analysis to identify patterns in multivariate datasets combining different aspects of CPK28 regulation

• Hierarchical clustering to identify groups of experimental conditions with similar CPK28 behavior

For all statistical analyses, appropriate biological and technical replicates (minimum n=3) should be included, and effect sizes alongside p-values should be reported to provide a complete picture of the biological significance of the observed differences.

How can researchers integrate CPK28 antibody data with other experimental approaches to build comprehensive models of plant immunity?

Integrating CPK28 antibody-derived data with other experimental approaches enables researchers to construct comprehensive models of plant immunity. Successful integration requires methodological diversity, incorporating genomic, transcriptomic, proteomic, and phenotypic data alongside antibody-based findings.

Researchers should combine CPK28 antibody data with genetic approaches by analyzing CPK28 functions in various genetic backgrounds, including mutants of interacting partners like BIK1, PUB25/26, ATL31/6, and PSKR1 . This genetic dissection helps establish epistatic relationships and pathway organization. For example, studying the PSK-induced growth promotion and disease susceptibility phenotypes in pskr1 cpk28 double mutants and OE-PSKR1 cpk28 lines has revealed that CPK28 functions downstream of PSKR1 in regulating the growth-defense balance .

Transcriptomic approaches should complement antibody-based protein studies. RNA-seq or microarray analyses comparing wild-type, cpk28 mutants, and CPK28 overexpression lines can identify genes and pathways regulated by CPK28. Additionally, comparing transcriptomes of plants expressing phospho-null (CPK28S318A) versus wild-type CPK28 can reveal phosphorylation-dependent transcriptional programs .

Advanced protein interaction studies using techniques like BioID, proximity labeling, or protein arrays combined with traditional co-immunoprecipitation using CPK28 antibodies provide comprehensive interactome data. These approaches have identified key interactors such as ATL31/6, which directly ubiquitinate CPK28 resulting in its proteasomal degradation .

Structural biology approaches provide critical insights into how phosphorylation affects CPK28 function. Intrinsic fluorescence experiments have demonstrated that Ser318 phosphorylation stabilizes the Ca²⁺-bound active state of CPK28, explaining how this modification primes CPK28 to respond to physiological calcium concentrations .

For systems-level integration, researchers should develop mathematical models incorporating CPK28 regulation and function. These models should account for:

• Dynamic changes in CPK28 abundance due to ubiquitination and proteasomal degradation

• Phosphorylation-dependent modulation of Ca²⁺ sensitivity

• Interactions with E3 ligases and substrate proteins

• Crosstalk with other signaling pathways

By integrating these diverse approaches, researchers can develop predictive models of how CPK28 contributes to immune homeostasis and the growth-defense balance in plants.

What emerging technologies could enhance the specificity and sensitivity of CPK28 antibody-based detection methods?

Emerging technologies offer promising avenues for enhancing CPK28 antibody specificity and sensitivity. Single-domain antibodies (nanobodies) derived from camelid heavy-chain antibodies represent a breakthrough technology applicable to CPK28 research. Their smaller size (approximately 15 kDa) enables access to epitopes that conventional antibodies cannot reach, potentially providing detection of specific CPK28 conformational states related to activation or interaction with partners . Their recombinant nature also ensures batch-to-batch consistency, addressing a common limitation of conventional antibodies.

CRISPR-epitope tagging strategies allow endogenous CPK28 to be tagged with small epitopes without disrupting its function. This approach enables detection using highly-specific commercial antibodies against tags like FLAG or HA, circumventing the need for developing specific anti-CPK28 antibodies while maintaining endogenous expression levels and regulation .

Proximity labeling technologies such as TurboID or APEX2 fused to CPK28 can biotinylate nearby proteins upon activation, allowing for streptavidin-based detection that is significantly more sensitive than conventional antibody approaches. This method is particularly valuable for detecting transient interactions that occur during immune signaling activation .

Mass spectrometry-based approaches, particularly selected reaction monitoring (SRM) or parallel reaction monitoring (PRM), offer antibody-independent quantification of CPK28 and its modifications. These methods can detect specific phosphorylated forms (such as pSer318) with high sensitivity and specificity, complementing antibody-based approaches .

Advanced microscopy techniques including super-resolution microscopy (STORM, PALM) combined with CPK28 antibodies can reveal nanoscale spatial organization of CPK28 at the plasma membrane and its dynamic redistribution during immune responses. Single-molecule tracking approaches using fluorescently-labeled antibody fragments can monitor CPK28 dynamics in live cells with unprecedented precision .

These emerging technologies, individually or in combination, will significantly enhance our ability to detect, quantify, and characterize CPK28 in complex biological samples, advancing our understanding of its regulatory mechanisms in plant immunity and growth.

How might CPK28 antibodies contribute to developing crops with enhanced disease resistance without compromising yield?

CPK28 antibodies can significantly contribute to agricultural biotechnology efforts aimed at developing crops with enhanced disease resistance while maintaining optimal yield. As a negative regulator of immunity and positive regulator of growth, CPK28 represents an ideal target for fine-tuning the growth-defense balance in crops .

Phospho-specific CPK28 antibodies targeting Ser318 can be utilized in high-throughput screening platforms to identify chemical compounds or bioactive molecules that modulate CPK28 phosphorylation status. Since Ser318 phosphorylation is specifically required for immune regulation but dispensable for growth promotion , compounds that inhibit this specific phosphorylation event could enhance plant immunity without compromising yield parameters.

For crop improvement programs, CPK28 antibodies serve as invaluable tools for phenotyping diverse germplasm. By analyzing CPK28 protein levels, phosphorylation status, and turnover rates across different cultivars using quantitative immunoblotting, researchers can identify genetic backgrounds with optimized CPK28 regulation. These natural variants could then be incorporated into breeding programs to develop cultivars with enhanced disease resistance and maintained yield potential.

Additionally, CPK28 antibodies can facilitate the characterization of transgenic crops expressing modified versions of CPK28. For instance, crops expressing CPK28S318A would theoretically exhibit enhanced disease resistance without growth penalties . CPK28 antibodies would be essential for confirming protein expression levels and assessing potential compensatory changes in the endogenous CPK28 regulatory network in these transgenic lines.

When developing such biotechnological applications, researchers must consider the conservation of CPK28 function across species. Phospho-specific antibodies can help determine whether Ser318 phosphorylation serves similar regulatory roles in crop species as observed in Arabidopsis . This cross-species validation is essential for successful translation of fundamental discoveries into agricultural applications.

Multi-year field trials of crops with modified CPK28 function will ultimately be required, with CPK28 antibodies serving as critical tools for monitoring protein dynamics under diverse environmental conditions and correlating molecular data with agronomically relevant traits such as disease resistance, growth parameters, and yield components.

What methodological improvements are needed to utilize CPK28 antibodies in studying the spatiotemporal dynamics of immune signaling?

Advancing our understanding of the spatiotemporal dynamics of immune signaling requires significant methodological improvements in CPK28 antibody applications. Current approaches often provide static snapshots of CPK28 status, failing to capture the dynamic nature of immune responses. To address this limitation, researchers need to develop live-cell imaging techniques compatible with CPK28 antibody-based detection.

One promising approach involves developing cell-permeable nanobodies or antibody fragments directed against CPK28 or its phosphorylated forms. These smaller antibody derivatives can be conjugated to fluorescent dyes or proteins and introduced into living plant cells via microinjection or cell-penetrating peptides, allowing real-time monitoring of CPK28 dynamics during immune responses .

Microfluidic systems combined with high-resolution microscopy offer another avenue for improvement. These systems can deliver precise doses of elicitors (such as flg22) while simultaneously imaging plant cells labeled with fluorescently-tagged CPK28 antibodies, enabling researchers to observe the immediate dynamics of CPK28 relocalization, degradation, or interaction with partners like ATL31/6 .

For tissue-level analyses, advances in clearing techniques and light-sheet microscopy can be combined with CPK28 immunolabeling to visualize protein distribution across entire plant organs in three dimensions. This approach would reveal how CPK28 regulation varies across different cell types and tissues during systemic immune responses.

Temporal resolution can be significantly improved through the development of automated sampling and fixation devices that can process plant tissues at precise intervals following immune elicitation. When combined with high-throughput immunoblotting or immunohistochemistry using CPK28 antibodies, these systems would generate detailed time-course data revealing the kinetics of CPK28 regulation across different subcellular compartments and cell types.

Additionally, multiplexed antibody labeling techniques need further development to simultaneously visualize CPK28 alongside its interaction partners and modifications. Advanced approaches like multiplexed ion beam imaging (MIBI) or co-detection by indexing (CODEX) could enable simultaneous detection of CPK28, its phosphorylated forms, and interaction partners like BIK1, PUB25/26, ATL31/6, and PSKR1 .

These methodological improvements would transform our understanding of CPK28 function from static models to dynamic frameworks that accurately capture the spatiotemporal complexity of plant immune signaling.