CPK13 Antibody

What is CPK13 and why is it significant in plant research?

CPK13 belongs to the calcium-dependent protein kinase (CPK) family in Arabidopsis thaliana, comprising 34 different genes. Unlike most CPKs, CPK13 is considered a calcium-insensitive type of CPK, functioning independently of calcium concentration changes . The significance of CPK13 lies in its specific expression in guard cells and its role in stomatal movement regulation. Research has demonstrated that CPK13 reduces stomatal aperture by inhibiting inward potassium channels KAT1 and KAT2, which are essential for stomatal opening .

The kinase activity of CPK13 has been experimentally confirmed to be independent of calcium, making it an unusual member of the CPK family. When overexpressed in Arabidopsis, CPK13 significantly reduces light-induced stomatal opening without affecting ABA-induced stomatal closure, suggesting it operates through a pathway independent of the hormone ABA . This unique regulatory mechanism makes CPK13 an important target for understanding stomatal physiology and potentially for engineering drought-resistant crops.

What types of CPK13 antibodies are available for research purposes?

While the search results don't specifically detail commercially available CPK13 antibodies, researchers typically use several types of antibodies for studying plant proteins like CPK13. These include:

• Polyclonal antibodies: Generated in animals (typically rabbits) immunized with purified CPK13 protein or synthetic peptides corresponding to unique regions of CPK13. These recognize multiple epitopes on the CPK13 protein.

• Monoclonal antibodies: Produced from a single B-cell clone and recognize a single epitope, offering high specificity for particular domains of CPK13.

• Tagged protein detection systems: For cases where generating specific antibodies is challenging, researchers often use tagged versions of CPK13 (with GST, GFP, etc.) and corresponding commercial antibodies against these tags.

Based on general antibody research practices seen in similar plant protein studies, antibodies against CPK13 would typically be optimized for applications including Western blotting, immunoprecipitation, immunolocalization, and potentially ELISA-based quantification .

How can I verify the specificity of a CPK13 antibody?

Verifying antibody specificity is crucial for reliable experimental results, particularly with plant CPKs that share sequence homology. A comprehensive verification protocol should include:

Positive and negative controls:

• Positive control: Using recombinant CPK13 protein or extracts from plants overexpressing CPK13, such as the OE13#3 and OE13#4 lines described in the literature .

• Negative control: Testing with extracts from CPK13 knockout plants (cpk13-) or testing with pre-immune serum.

Cross-reactivity testing:

• Western blot analysis with recombinant proteins of closely related CPKs to ensure the antibody does not cross-react with other family members.

• Competitive binding assays using CPK13 peptides to block antibody binding.

Experimental validation:

• Immunoprecipitation followed by mass spectrometry to confirm pull-down of CPK13.

• Comparing immunolocalization patterns with GFP-tagged CPK13 expression patterns.

• Preabsorption tests using the immunizing peptide to confirm specificity.

What are the optimal protocols for using CPK13 antibodies in Western blot analysis?

For optimal Western blot detection of CPK13 in plant samples, researchers should consider the following protocol based on successful approaches with similar plant proteins:

Sample preparation:

• Extract total proteins from guard cell-enriched epidermal peels to maximize CPK13 detection, as CPK13 is predominantly expressed in guard cells .

• Use a buffer containing phosphatase inhibitors to preserve phosphorylation states.

• For comprehensive analysis, separate nuclear and cytosolic fractions to assess CPK13 distribution.

SDS-PAGE and transfer conditions:

• Use 10-12% acrylamide gels for optimal separation (CPK13 is approximately 55-60 kDa).

• Transfer proteins to PVDF membranes at 100V for 60-90 minutes in cold transfer buffer containing 10-20% methanol.

Blocking and antibody incubation:

• Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Incubate with primary CPK13 antibody at 1:1000 to 1:5000 dilution (optimization required) overnight at 4°C.

• Wash thoroughly with TBST buffer (3-5 washes, 5-10 minutes each).

• Incubate with appropriate secondary antibody conjugated to HRP at 1:5000 to 1:10000 dilution for 1 hour at room temperature.

Detection strategies:

• Use enhanced chemiluminescence (ECL) for standard detection.

• For quantitative analysis, consider fluorescently-labeled secondary antibodies and an imaging system that allows direct quantification.

Validation controls:

• Include extracts from wild-type plants, CPK13 overexpression lines (OE13), and CPK13 knockout lines when available.

• Use anti-GST antibody if detecting recombinant GST-tagged CPK13 as shown in Figure 1A of the research .

How can CPK13 antibodies be utilized for co-immunoprecipitation to study protein interactions?

CPK13 has been shown to interact with potassium channels KAT1 and KAT2 in guard cells . Researchers interested in studying these or other protein interactions can utilize CPK13 antibodies for co-immunoprecipitation (Co-IP) following these methodological guidelines:

Sample preparation:

• Harvest tissue with confirmed CPK13 expression (guard cell-enriched epidermal peels).

• Grind tissue in liquid nitrogen and extract proteins using a non-denaturing lysis buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% NP-40 or 0.5% Triton X-100

  • Protease inhibitor cocktail

  • Phosphatase inhibitors (to preserve phosphorylation states)

Immunoprecipitation procedure:

• Pre-clear lysate with Protein A/G beads to reduce non-specific binding.

• Incubate cleared lysate with CPK13 antibody (2-5 μg per 1 mg of total protein) overnight at 4°C with gentle rotation.

• Add Protein A/G beads and incubate for 2-4 hours at 4°C.

• Wash beads 4-5 times with cold lysis buffer.

• Elute proteins by boiling in SDS sample buffer or using mild elution conditions if maintaining protein activity is important.

Interaction analysis:

• Analyze co-precipitated proteins by SDS-PAGE followed by Western blotting with antibodies against suspected interaction partners (e.g., KAT1, KAT2).

• Consider mass spectrometry analysis of co-precipitated proteins for unbiased discovery of novel interaction partners.

Alternative validation approaches:

• Perform reciprocal Co-IP using antibodies against the interaction partner.

• Validate interactions using alternative methods such as the FRET-FLIM approach that was successfully used to demonstrate CPK13-KAT2 interaction in planta .

What immunohistochemistry protocols work best for localizing CPK13 in plant tissues?

For successful immunohistochemical localization of CPK13 in plant tissues, particularly in guard cells where it is predominantly expressed, researchers should consider this protocol:

Tissue fixation and preparation:

• Fix fresh tissue samples in 4% paraformaldehyde in PBS (pH 7.4) for 4-6 hours at 4°C.

• Wash with PBS buffer (3 times, 10 minutes each).

• Dehydrate through an ethanol series (30%, 50%, 70%, 95%, 100%).

• Embed in paraffin or resin depending on the desired section thickness.

• Section tissues to 5-10 μm thickness.

Immunostaining procedure:

• Deparaffinize sections and rehydrate through a descending ethanol series.

• Perform antigen retrieval if necessary (citrate buffer pH 6.0, microwave heating).

• Block endogenous peroxidase activity with 3% hydrogen peroxide if using HRP-based detection.

• Block non-specific binding with 5% normal serum from the species of the secondary antibody.

• Incubate with primary CPK13 antibody (1:100 to 1:500 dilution) overnight at 4°C.

• Wash thoroughly with PBS (3 times, 5 minutes each).

• Incubate with appropriate labeled secondary antibody for 1-2 hours at room temperature.

• Wash thoroughly with PBS.

• For fluorescent detection, counterstain with DAPI to visualize nuclei.

• Mount slides with anti-fade mounting medium.

Controls and validation:

• Include negative controls by omitting primary antibody or using pre-immune serum.

• Use CPK13 knockout tissue as a negative control when available.

• Compare immunostaining patterns with CPK13 promoter-GUS expression patterns as shown in Figure 1C from the research .

• Consider dual immunolabeling with antibodies against known guard cell markers to confirm cell-type specificity.

How can phospho-specific antibodies be developed to study CPK13 activity and substrate phosphorylation?

Developing phospho-specific antibodies to study CPK13-mediated phosphorylation events requires sophisticated approaches:

Target phosphorylation site identification:

• Analyze the literature for known CPK13 phosphorylation sites on substrates like KAT1 and KAT2 channels.

• Conduct in vitro kinase assays using recombinant CPK13 and substrate peptides to identify phosphorylation sites.

• Use phosphopeptide arrays, as described in the research, where CPK13 was shown to phosphorylate specific peptides derived from KAT1 and KAT2 .

• Confirm phosphorylation sites using mass spectrometry.

Phospho-specific antibody production:

• Design phosphopeptides containing the identified phosphorylation site with the following considerations:

  • 10-15 amino acids in length with the phosphorylated residue centrally located

  • Conjugation to carrier protein (KLH or BSA) for immunization

  • Prepare both phosphorylated and non-phosphorylated versions of the peptide

• Immunize rabbits or other suitable animals with the phosphopeptide-carrier conjugate.

• Collect antiserum and purify using a two-step affinity purification:

  • First, pass through a column with immobilized non-phosphopeptide to remove antibodies recognizing the non-phosphorylated epitope

  • Then, affinity-purify phospho-specific antibodies using a column with the immobilized phosphopeptide

Validation of phospho-specific antibodies:

• Test antibody specificity using dot blots or ELISA with phosphorylated and non-phosphorylated peptides.

• Validate using Western blotting with:

  • In vitro phosphorylated substrates (with and without phosphatase treatment)

  • Extracts from wild-type plants and CPK13 overexpression lines

  • Controls including phosphatase-treated samples and samples from plants expressing phospho-mutant versions of the substrate

Research applications of phospho-specific antibodies:

• Monitor phosphorylation status of CPK13 substrates under different physiological conditions.

• Assess spatial distribution of phosphorylated substrates using immunohistochemistry.

• Quantify phosphorylation levels in response to environmental stimuli.

What are the best approaches to study CPK13 localization and dynamics using antibody-based techniques?

Studying CPK13 localization and dynamics in plant cells requires sophisticated antibody-based approaches:

Immunofluorescence microscopy:

• Prepare plant samples using the previously described immunohistochemistry protocol.

• Use high-resolution confocal microscopy to determine subcellular localization in guard cells.

• Perform co-localization studies with markers for different cellular compartments.

• Use deconvolution or super-resolution microscopy techniques for detailed subcellular localization.

Live cell imaging approaches:

• Generate transgenic plants expressing CPK13 fused to a fluorescent protein (FP) as demonstrated in the FRET-FLIM experiments described in the research .

• Validate the functionality of the fusion protein by complementation of CPK13 knockout phenotypes.

• For antibody-based tracking in live cells, consider using:

  • Membrane-permeable fluorescently labeled nanobodies against FP-tagged CPK13

  • Microinjection of labeled anti-CPK13 antibody fragments

Temporal dynamics studies:

• Use inducible expression systems to monitor CPK13 expression and localization over time.

• Combine with calcium imaging to correlate CPK13 activity with calcium signals, despite CPK13 being calcium-insensitive.

• Track CPK13 movement in response to stimuli known to affect stomatal aperture.

Advanced microscopy techniques:

• Förster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) as used in the referenced research to study CPK13-KAT2 interactions .

• Fluorescence Recovery After Photobleaching (FRAP) to study CPK13 mobility.

• Bimolecular Fluorescence Complementation (BiFC) to visualize protein-protein interactions in vivo.

Table 1: Comparison of Methods for Studying CPK13 Localization and Dynamics

How can CPK13 antibodies be utilized to investigate calcium-independent regulation mechanisms?

CPK13 provides a unique model to study calcium-independent regulation in a protein family generally known for calcium dependence . Antibodies against CPK13 can be valuable tools to investigate these mechanisms:

Comparative analysis with calcium-dependent CPKs:

• Develop a panel of antibodies against different CPK family members, including strictly Ca²⁺-dependent, Ca²⁺-stimulated, and Ca²⁺-insensitive CPKs like CPK13.

• Perform immunoprecipitation of different CPKs followed by:

  • In vitro kinase assays at varying calcium concentrations

  • Structural analysis using limited proteolysis to detect conformation changes

  • Mass spectrometry to identify post-translational modifications and interacting partners

Investigating regulatory domains:

• Generate domain-specific antibodies targeting:

  • N-terminal variable domain

  • Kinase domain

  • Autoinhibitory domain

  • Calmodulin-like domain

• Use these antibodies to examine conformational changes and domain interactions in the presence/absence of calcium.

• Compare immunoprecipitation results between wild-type CPK13 and mutated versions with alterations in calcium-binding domains.

Experimental approach to identify calcium-independent regulation mechanisms:

• Immunoprecipitate CPK13 from plants under different stress conditions known to affect stomatal regulation.

• Analyze post-translational modifications by mass spectrometry.

• Identify interacting proteins that might regulate CPK13 activity in a calcium-independent manner.

• Compare phosphorylation targets in vitro using the peptide array approach as described in the research .

Correlation with physiological responses:

• Monitor CPK13 protein levels, phosphorylation status, and activity in response to environmental stimuli that trigger stomatal movements.

• Compare these responses between wild-type plants and calcium signaling mutants.

• Correlate findings with stomatal aperture measurements and potassium channel activity as demonstrated in the research where overexpression of CPK13 reduced stomatal aperture .

What are the common challenges in detecting CPK13 in plant samples and how can they be overcome?

Researchers working with plant proteins like CPK13 often encounter specific challenges that require optimization:

Challenge 1: Low abundance of CPK13 in whole-plant extracts
Solution approaches:

• Enrich for guard cells using epidermal peels or guard cell protoplast isolation since CPK13 is predominantly expressed in guard cells .

• Use immunoprecipitation to concentrate CPK13 before Western blot analysis.

• Consider using more sensitive detection methods such as chemiluminescent substrates with enhanced sensitivity or amplification systems.

• Optimize protein extraction using buffers containing chaotropic agents (urea, thiourea) for better solubilization.

Challenge 2: Cross-reactivity with other CPK family members
Solution approaches:

• Design peptide antigens from unique regions of CPK13, avoiding conserved kinase and calcium-binding domains.

• Perform antibody pre-absorption with recombinant proteins of closely related CPKs.

• Use CPK13 knockout plants as negative controls to identify non-specific bands.

• Consider using epitope-tagged CPK13 in transgenic plants if specific antibodies cannot be generated.

Challenge 3: Post-translational modifications affecting antibody recognition
Solution approaches:

• Generate antibodies against multiple epitopes throughout the CPK13 protein.

• Prevent phosphatase activity during extraction by including phosphatase inhibitors in extraction buffers.

• Compare detection patterns under different physiological conditions that might alter post-translational modifications.

Challenge 4: Interference from plant compounds
Solution approaches:

• Add polyvinylpolypyrrolidone (PVPP) to extraction buffers to remove phenolic compounds.

• Include higher concentrations of reducing agents (DTT, β-mercaptoethanol) to prevent oxidation.

• Use TCA/acetone precipitation to clean up samples before SDS-PAGE.

• Consider plant-optimized protein extraction kits designed to remove interfering compounds.

How can CPK13 antibodies be optimized for studying the CPK13-KAT2 channel regulatory mechanism?

The research has established that CPK13 phosphorylates and inhibits KAT2 potassium channels in guard cells . To further investigate this regulatory mechanism using antibodies:

Co-localization studies:

• Perform dual immunolabeling with anti-CPK13 and anti-KAT2 antibodies in guard cells.

• Use high-resolution confocal or super-resolution microscopy to determine if co-localization occurs in specific membrane microdomains.

• Compare localization patterns in wild-type plants versus plants under conditions that affect stomatal aperture.

In situ proximity ligation assay (PLA):

• Use primary antibodies against CPK13 and KAT2, followed by oligonucleotide-conjugated secondary antibodies.

• When the two proteins are in close proximity (<40 nm), the oligonucleotides can interact, allowing rolling circle amplification and detection.

• This technique provides spatial information about CPK13-KAT2 interactions in intact plant tissues.

Phosphorylation site analysis:

• Based on the peptide array data showing CPK13 phosphorylation of specific KAT2 peptides , develop phospho-specific antibodies against these sites.

• Use these antibodies to monitor KAT2 phosphorylation status in:

  • Wild-type plants versus CPK13 overexpression lines

  • Plants under different light conditions that affect stomatal opening

  • Plants treated with kinase inhibitors

Functional correlation studies:

• Correlate the level of CPK13-KAT2 interaction (measured by Co-IP or PLA) with:

  • KAT2 phosphorylation status (using phospho-specific antibodies)

  • Inward K⁺ channel activity (measured by electrophysiology)

  • Stomatal aperture measurements

• Investigate how environmental factors affecting stomatal movements influence these parameters.

Table 2: CPK13-Mediated Regulation of KAT2 Channels - Experimental Approaches

How can new antibody technologies enhance CPK13 research in plant science?

Emerging antibody technologies offer significant potential to advance CPK13 research beyond conventional approaches:

Single-domain antibodies (nanobodies):

• Develop camelid-derived single-domain antibodies (VHHs) or shark-derived IgNARs against CPK13.

• Advantages for plant research include:

  • Smaller size allowing better tissue penetration

  • Stability under reducing conditions found in plant cells

  • Potential for intracellular expression as "intrabodies"

• Applications include intracellular tracking of CPK13 in living plant cells and modulation of CPK13 function through intrabody expression.

Recombinant antibody fragments:

• Engineer Fab, scFv, or other antibody fragments optimized for plant research.

• Create bi-specific antibody formats that can simultaneously bind CPK13 and its interaction partners or substrates.

• Develop antibody-based biosensors to monitor CPK13 activity in real-time.

Antibody-based proximity labeling:

• Fuse CPK13 antibodies or binding fragments to promiscuous biotin ligases (BioID, TurboID) or peroxidases (APEX).

• Use these conjugates to identify proteins in close proximity to CPK13 in intact cells through biotinylation of nearby proteins.

• This approach can reveal the CPK13 interactome in specific cellular contexts or under different physiological conditions.

Optogenetic applications:

• Develop photoactivatable antibody fragments against CPK13 that change binding properties upon light stimulation.

• Use these tools to manipulate CPK13 activity with spatial and temporal precision in planta.

• Combine with electrophysiological recordings to directly link CPK13 inhibition to K⁺ channel activity.

What are the methodological considerations for studying CPK13 antibody specificity across different plant species?

CPK13 research could extend beyond Arabidopsis to crops or other model plant species. When using CPK13 antibodies across species, researchers should consider:

Sequence conservation analysis:

• Perform bioinformatic analysis of CPK13 homologs across target plant species.

• Identify conserved and variable regions that might affect antibody recognition.

• Align sequences of the epitope regions used for antibody generation across species.

Cross-reactivity testing protocol:

• Express recombinant CPK13 proteins from different plant species.

• Perform Western blot analysis to assess antibody cross-reactivity.

• Optimize antibody concentration and washing conditions for each species.

• Consider developing a panel of antibodies targeting different epitopes to increase the likelihood of cross-species recognition.

Validation in diverse plant materials:

• Test antibodies in:

  • Wild-type plants of target species

  • Transgenic plants with altered CPK13 expression when available

  • Plants under conditions known to affect CPK13 expression or activity

• Compare immunolocalization patterns with in situ hybridization results or promoter-reporter studies.

Epitope mapping considerations:

• For polyclonal antibodies showing partial cross-reactivity, perform epitope mapping to identify which epitopes are recognized across species.

• Consider affinity purification against conserved epitopes to enrich for antibodies with cross-species utility.

• For critical applications, develop species-specific antibodies against unique regions of CPK13 homologs.

Table 3: Predicted Cross-Reactivity of Arabidopsis CPK13 Antibodies with Related Plant Species