CIPK14 Antibody

What is CIPK14 and why is it significant in plant research?

CIPK14 (CBL-interacting protein kinase 14) functions as an essential component in calcium-mediated signal transduction pathways in plants. It plays a critical role as a negative regulator in plant immune responses, particularly in Arabidopsis. Research has demonstrated that CIPK14 loss-of-function mutants exhibit enhanced resistance to Pseudomonas syringae, while overexpression plants show increased susceptibility to bacterial pathogens . The enhanced resistance in cipk14 mutants correlates with increased salicylic acid (SA) accumulation and elevated expression of defense marker genes including PR1, EDS1, EDS5, and ICS1 . This kinase's regulatory role extends to both transcriptional and post-transcriptional levels, making it a significant target for understanding plant immunity mechanisms.

What types of antibodies are typically used for CIPK14 detection?

For CIPK14 detection, researchers typically employ three main types of antibodies:

• Polyclonal antibodies: Generated against multiple epitopes of the CIPK14 protein, offering broad recognition but potentially lower specificity.

• Monoclonal antibodies: Produced against a single epitope, providing high specificity but potentially limited in detecting all protein variants.

• Phospho-specific antibodies: Designed to recognize phosphorylated forms of CIPK14, particularly important since CIPK14 functions through phosphorylation of targets like WHY1 .

When selecting an antibody, researchers should consider whether they need to detect total CIPK14 protein or specific phosphorylated states, as this will determine which antibody type is most appropriate for experimental goals.

How do I design an experiment to study CIPK14 interactions with WHY1/WHY3 proteins?

Designing experiments to study CIPK14 interactions with WHY1/WHY3 proteins requires a multi-faceted approach:

• Co-immunoprecipitation (Co-IP): Use anti-CIPK14 antibodies to pull down protein complexes, followed by Western blotting with anti-WHY1/WHY3 antibodies to detect interactions.

• In vitro kinase assays: Assess phosphorylation of WHY1 by CIPK14 using purified proteins, followed by detection with phospho-specific antibodies or radioactive labeling.

• Subcellular localization studies: Compare the distribution of WHY1 between plastids and nucleus in wild-type versus cipk14 mutants using fractionation and immunoblotting.

• Proteomic comparison: Analyze differential protein expression between wild-type, why1/3 mutants, and CIPK14 overexpression lines as demonstrated in previous research .

A comprehensive experimental design should include appropriate controls:

• Wild-type plants

• CIPK14 knockout mutants (cipk14)

• CIPK14 overexpression lines (oeCIPK14)

• WHY1/WHY3 knockout mutants (why1/3)

• WHY1 overexpression lines (oeWHY1)

This approach has successfully identified that CIPK14 interacts with and phosphorylates WHY1, affecting its distribution between cellular compartments .

What are the essential validation steps for a CIPK14 antibody?

Validating a CIPK14 antibody requires several critical steps to ensure specificity, selectivity, and reproducibility:

• Specificity testing:

  • Western blot analysis using wild-type plant tissues alongside cipk14 knockout mutants

  • Testing against recombinant CIPK14 protein

  • Peptide competition assays to confirm epitope specificity

• Cross-reactivity assessment:

  • Testing against closely related CIPK family members

  • Evaluation in multiple plant species if cross-species reactivity is claimed

• Application-specific validation:

  • For immunohistochemistry: Comparison of staining patterns between wild-type and cipk14 mutants

  • For immunoprecipitation: Verification of pulled-down proteins by mass spectrometry

  • For immunofluorescence: Co-localization with known markers of expected cellular compartments

• Reproducibility testing:

  • Analysis across different antibody lots

  • Comparison with alternative validated antibodies targeting the same protein

These validation steps align with the FDA's definition of validation as "the process of demonstrating, through the use of specific laboratory investigations, that the performance characteristics of an analytical method are suitable for its intended analytical use" .

How can I distinguish between specific and non-specific binding in CIPK14 antibody applications?

Distinguishing between specific and non-specific binding requires a systematic approach with appropriate controls:

• Negative controls:

  • Use cipk14 knockout plant tissues where CIPK14 protein is absent

  • Include no-primary-antibody controls in immunoassays

  • Employ pre-immune serum controls for polyclonal antibodies

• Blocking peptide competition:

  • Pre-incubate the CIPK14 antibody with excess specific peptide antigen

  • Compare results with and without peptide competition

  • Specific binding should be significantly reduced or eliminated with peptide competition

• Signal verification methods:

  • Use multiple antibodies targeting different epitopes of CIPK14

  • Confirm protein identity with mass spectrometry after immunoprecipitation

  • Correlate antibody detection with transcript levels using qRT-PCR

• Band size verification:

  • Ensure detected protein matches the predicted molecular weight of CIPK14

  • For post-translationally modified forms, verify band shifts correspond to expected modifications

When determining specificity, remember that non-specific binding typically appears as multiple unexpected bands or diffuse staining patterns that don't correspond to known expression patterns of CIPK14.

What quantitative methods can assess CIPK14 antibody sensitivity and dynamic range?

Several quantitative methods can effectively assess CIPK14 antibody sensitivity and dynamic range:

• Serial dilution analysis:

  • Create standard curves using purified recombinant CIPK14 protein

  • Plot signal intensity versus protein concentration

  • Determine lower limit of detection (LLOD) and limit of quantification (LOQ)

• Dynamic range assessment:

  • Compare signal from samples with varying CIPK14 expression levels

  • Include wild-type, cipk14 mutants, and CIPK14 overexpression lines

  • Quantify linearity across concentration ranges

• Signal-to-noise ratio (SNR) calculation:

  • Measure specific signal intensity versus background

  • SNR > 3 typically indicates reliable detection

  • Calculate Z-factor to assess assay quality: Z = 1 - (3σc+ + 3σc-)/|μc+ - μc-|

• Comparative analysis with reference methods:

  • Correlate antibody-based quantification with mass spectrometry data

  • Compare results with transcript quantification by qRT-PCR

  • Validate across multiple detection platforms (western blot, ELISA, immunofluorescence)

For consistent quantitation, establish internal standards and include them in each experiment to normalize between assays and account for experimental variation.

How should I optimize immunoblotting protocols for CIPK14 detection in plant tissues?

Optimizing immunoblotting protocols for CIPK14 detection requires attention to several key parameters:

• Sample preparation:

  • Use extraction buffers containing phosphatase inhibitors to preserve phosphorylation states

  • Include protease inhibitors to prevent degradation

  • Fresh tissue extraction is preferable to frozen samples for preserving protein integrity

• Protein separation:

  • Use 10-12% SDS-PAGE gels for optimal resolution of CIPK14 (expected MW ~45-55 kDa)

  • For phosphorylated CIPK14 detection, consider Phos-tag gels to enhance separation of phosphorylated forms

• Transfer optimization:

  • Semi-dry transfer for 1 hour or wet transfer overnight at low voltage

  • PVDF membranes generally provide better results than nitrocellulose for kinases

• Blocking and antibody incubation:

  • Test both BSA and non-fat dry milk as blocking agents (kinase detection often works better with BSA)

  • Optimize primary antibody dilution (typically 1:500 to 1:2000)

  • Extended incubation at 4°C overnight often improves specific signal

• Signal development:

  • ECL-based detection for standard applications

  • Consider fluorescence-based detection for more precise quantification

• Critical controls:

  • Include wild-type, cipk14 mutant, and CIPK14 overexpression samples

  • Use loading controls such as anti-actin or anti-tubulin antibodies

  • For phospho-specific detection, include samples treated with phosphatase

This optimized approach has been successfully implemented in studies examining CIPK14's role in plant immunity and its interaction with WHY proteins .

What are the best practices for using CIPK14 antibodies in immunoprecipitation studies?

For successful immunoprecipitation (IP) of CIPK14 and its interacting partners, follow these best practices:

• Lysate preparation:

  • Use mild, non-denaturing lysis buffers to preserve protein-protein interactions

  • Include both phosphatase and protease inhibitors

  • Clear lysate by centrifugation at high speed before IP

• Antibody selection and coupling:

  • Choose antibodies raised against regions not involved in protein-protein interactions

  • Consider covalently coupling antibodies to beads to avoid heavy/light chain interference in subsequent detection

  • For co-IP studies, verify that the antibody doesn't interfere with CIPK14-WHY1 interaction

• IP procedure optimization:

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

  • Optimize antibody-to-lysate ratio (typically 2-5 μg antibody per mg of total protein)

  • Include appropriate negative controls (non-specific IgG, cipk14 knockout samples)

• Washing conditions:

  • Balance stringency of washes to remove non-specific interactions while preserving specific ones

  • Consider testing increasing salt concentrations to determine optimal washing conditions

  • Include 0.1-0.5% mild detergent (e.g., NP-40) in wash buffers

• Elution and detection:

  • Elute under native conditions for functional studies of immunoprecipitated complexes

  • For protein identification, analysis by mass spectrometry is preferable to Western blotting alone

  • Perform reverse IP (using antibodies against suspected interacting partners) to confirm interactions

This approach has been instrumental in confirming that CIPK14 interacts with and phosphorylates WHY1, affecting its subcellular distribution and function in plant defense mechanisms .

How can I use CIPK14 antibodies to study its subcellular localization and translocation?

To study CIPK14 subcellular localization and potential translocation events:

• Immunofluorescence microscopy:

  • Fix plant tissues with paraformaldehyde while preserving cellular structures

  • Optimize permeabilization conditions for different cellular compartments

  • Use fluorescence-conjugated secondary antibodies with minimal cross-reactivity

  • Include co-staining with compartment-specific markers (nuclear, cytoplasmic, membrane)

• Subcellular fractionation:

  • Separate cellular components (nuclear, cytosolic, membrane, plastid fractions)

  • Perform Western blotting on each fraction with anti-CIPK14 antibodies

  • Include fraction-specific markers to confirm separation quality

  • Quantify CIPK14 distribution across fractions under different conditions or treatments

• Proximity ligation assay (PLA):

  • Detect interactions between CIPK14 and partners like WHY1 in situ

  • Visualize specific interaction sites within cellular compartments

  • Quantify interaction events in different cellular locations

• Live cell imaging with fusion proteins:

  • Validate antibody-based localization studies with GFP-CIPK14 constructs

  • Compare antibody staining patterns with fluorescent protein localization

  • Study dynamic changes in localization using inducible expression systems

Research has shown that CIPK14 affects the distribution of WHY1 between plastids and the nucleus through phosphorylation . When designing localization studies, include experiments that can detect potential changes in CIPK14 localization in response to calcium signaling or pathogen challenge, as these may be critical to understanding its function in plant immunity.

How do I troubleshoot weak or absent signal when using CIPK14 antibodies?

When faced with weak or absent signals when using CIPK14 antibodies, systematically address these potential issues:

• Protein extraction and preservation:

  • Verify protein extraction efficiency with total protein stains

  • Ensure use of fresh protease inhibitors in extraction buffers

  • Minimize freeze-thaw cycles of protein samples

  • Test different extraction buffers optimized for membrane-associated proteins

• Antibody-related factors:

  • Verify antibody quality with positive control samples

  • Try different antibody concentrations (perform a titration experiment)

  • Test alternative CIPK14 antibodies targeting different epitopes

  • Consider antibody storage conditions and avoid repeated freeze-thaw cycles

• Detection system optimization:

  • Increase antibody incubation time (overnight at 4°C)

  • Try more sensitive detection systems (enhanced chemiluminescence plus)

  • Reduce washing stringency while maintaining specificity

  • For fluorescence-based detection, adjust scanner settings and exposure times

• Expression level considerations:

  • CIPK14 may be expressed at low levels in certain tissues or conditions

  • Use tissues where CIPK14 is known to be expressed (based on transcriptome data)

  • Consider enrichment by immunoprecipitation before detection

  • Include CIPK14 overexpression samples as positive controls

• Epitope accessibility:

  • Test different protein denaturation conditions

  • For immunohistochemistry, optimize antigen retrieval methods

  • Consider native vs. reducing conditions for epitope exposure

If troubleshooting attempts fail to improve detection, consider alternative methods such as mass spectrometry-based approaches to detect and quantify CIPK14 protein.

What factors might cause contradictory results between CIPK14 protein and transcript levels?

Several factors can explain contradictions between CIPK14 protein levels (detected by antibodies) and transcript levels (measured by qRT-PCR):

• Post-transcriptional regulation:

  • microRNA-mediated regulation affecting translation efficiency

  • RNA binding proteins controlling transcript stability

  • Alternative splicing generating protein isoforms not recognized by the antibody

• Post-translational mechanisms:

  • Protein stability differences (degradation rates varying between conditions)

  • Proteasomal degradation targeting specific protein pools

  • Sequestration in different cellular compartments affecting extraction efficiency

• Technical considerations:

  • Antibody specificity issues or detection of only specific protein forms

  • Protein extraction bias for certain cellular compartments

  • qRT-PCR primer specificity for particular transcript variants

• Biological timing:

  • Temporal delay between transcription and translation

  • Different half-lives of mRNA versus protein

  • Feedback loops regulating protein but not transcript levels

Research on CIPK14 and WHY1/3 has demonstrated this phenomenon, with only 5 out of 26 genes showing consistent patterns between transcript and protein expression levels . This discrepancy highlights the importance of integrating both transcriptomic and proteomic approaches when studying CIPK14 function.

This data emphasizes the necessity of protein-level studies using validated antibodies rather than relying solely on transcript analysis.

How can I correctly interpret changes in CIPK14 phosphorylation status using phospho-specific antibodies?

Interpreting changes in CIPK14 phosphorylation status requires careful consideration of several aspects:

• Validation of phospho-specific antibodies:

  • Confirm antibody specificity using phosphatase-treated samples as negative controls

  • Verify recognition of phospho-mimetic mutants versus phospho-deficient mutants

  • Include in vitro phosphorylated CIPK14 as a positive control

• Quantification approach:

  • Always normalize phospho-signal to total CIPK14 protein

  • Use dual detection with different fluorophores for simultaneous measurement

  • Present data as phospho-CIPK14/total CIPK14 ratio rather than absolute values

• Dynamic analysis:

  • Monitor phosphorylation changes over a time course following stimulus

  • Consider multiple phosphorylation sites and their potential interdependence

  • Compare phosphorylation patterns in different subcellular compartments

• Functional correlation:

  • Relate phosphorylation changes to downstream events (e.g., WHY1 localization)

  • Correlate phosphorylation status with CIPK14 kinase activity in vitro

  • Compare phosphorylation patterns between wild-type and mutant plants with altered phenotypes

• Controls for biological significance:

  • Include treatments with calcium signaling modulators

  • Compare pathogen-challenged versus non-challenged samples

  • Analyze phosphorylation in plants with altered CBL (Calcineurin B-like) expression

This approach has been valuable in understanding how CIPK14-mediated phosphorylation affects WHY1 distribution between plastids and the nucleus, influencing plant defense responses . Changes in phosphorylation status of CIPK14 targets like MPK3/6 under flg22 treatment suggest that CIPK14 functions as a modulator of plant immunity at both transcriptional and post-transcriptional levels .

How do CIPK14 antibodies perform in cross-species applications?

The performance of CIPK14 antibodies across different plant species requires careful evaluation:

• Epitope conservation analysis:

  • Perform sequence alignment of CIPK14 homologs across species

  • Focus on antibody epitope regions to predict cross-reactivity

  • Generate conservation scores for epitope regions compared to full protein

• Validation strategy for cross-species use:

  • Test antibodies on recombinant CIPK14 proteins from target species

  • Include appropriate positive controls (overexpression) and negative controls (knockout if available)

  • Perform Western blotting with increasing protein amounts to determine sensitivity thresholds

• Performance characteristics by species:

  • Expect stronger signals in closely related species (within same family)

  • Adjust antibody concentrations for more distant species

  • Be prepared for potential cross-reactivity with other CIPK family members

• Application-specific considerations:

  • Western blotting typically shows better cross-species reactivity than immunohistochemistry

  • For immunoprecipitation, binding efficiency may vary significantly between species

  • In microscopy applications, background issues may be more prominent in non-model species

When using CIPK14 antibodies developed against Arabidopsis in other species, validation should include comparison with known expression patterns and molecular weight verification, as post-translational modifications might differ across species.

What advanced techniques combine CIPK14 antibodies with other molecular approaches for functional studies?

Integrating CIPK14 antibodies with advanced molecular techniques creates powerful approaches for functional studies:

• ChIP-seq (Chromatin Immunoprecipitation with sequencing):

  • If CIPK14 has nuclear functions, ChIP-seq can identify genomic binding regions

  • Requires highly specific antibodies capable of efficient immunoprecipitation

  • Can reveal CIPK14's role in transcriptional regulation if it associates with chromatin

• IP-MS (Immunoprecipitation coupled with Mass Spectrometry):

  • Identify novel CIPK14 interacting partners beyond known ones like WHY1

  • Detect post-translational modifications on CIPK14 and its substrates

  • Compare interactomes under different stress conditions or developmental stages

• Proximity-dependent labeling:

  • Fuse CIPK14 with BioID or APEX2 and validate localization with antibodies

  • Map spatial proteomics of CIPK14 neighborhood in different cellular compartments

  • Identify transient interactions missed by conventional co-IP approaches

• Single-cell analysis:

  • Combine immunofluorescence with single-cell transcriptomics

  • Correlate CIPK14 protein levels with cell-specific responses

  • Reveal cell-type specific functions in heterogeneous tissues

• CRISPR-edited systems with antibody validation:

  • Generate epitope-tagged endogenous CIPK14 using CRISPR

  • Compare antibody detection of native versus tagged protein

  • Study function in physiological context without overexpression artifacts

These integrated approaches can address complex questions about CIPK14 function, such as how its phosphorylation of WHY1 affects nuclear-plastid communication and ultimately regulates plant immunity .

How can I use CIPK14 antibodies to study its role in calcium-dependent signaling networks?

To investigate CIPK14's function in calcium-dependent signaling networks:

• Co-immunoprecipitation of signaling complexes:

  • Use CIPK14 antibodies to pull down intact signaling complexes

  • Identify CBL (Calcineurin B-like) partners that regulate CIPK14

  • Compare complex composition under different calcium concentrations

• Calcium ionophore and chelator studies:

  • Treat plants with calcium ionophores (A23187) or chelators (EGTA)

  • Monitor CIPK14 phosphorylation state and localization changes using specific antibodies

  • Track downstream substrate phosphorylation (such as WHY1) in response to calcium flux

• In situ activation analysis:

  • Combine calcium imaging (using indicators like Fluo-4) with CIPK14 immunolocalization

  • Create temporal maps of calcium signals and subsequent CIPK14 redistribution

  • Correlate with activation of defense responses in pathogen-challenged tissues

• Pathway reconstruction:

  • Immunoprecipitate CIPK14 from wild-type and mutant plants lacking specific signaling components

  • Perform in vitro kinase assays to assess how pathway perturbations affect CIPK14 activity

  • Use phospho-specific antibodies to monitor activation status of CIPK14 and its substrates

• Quantitative signaling dynamics:

  • Develop phospho-flow cytometry approaches using CIPK14 phospho-specific antibodies

  • Measure signaling dynamics across populations of cells or protoplasts

  • Create mathematical models of calcium-CIPK14 signaling incorporating quantitative antibody-based measurements

Research has established that CIPK14 functions in calcium-mediated signal transduction pathways and plays important roles in plant immunity . Understanding its activation and regulation in response to calcium signals will provide insights into how plants transduce external stimuli into appropriate defense responses.

How should I design experiments to compare CIPK14 with other CIPK family members?

When designing experiments to compare CIPK14 with other CIPK family members:

• Antibody specificity assessment:

  • Test cross-reactivity of CIPK14 antibodies with recombinant proteins of related CIPKs

  • Create epitope maps to identify unique regions for generating specific antibodies

  • Include multiple CIPK knockout lines as specificity controls

• Expression pattern comparison:

  • Design systematic tissue profiling using validated antibodies for each CIPK

  • Use consistent extraction and detection methods across all family members

  • Create standardized quantification approaches normalizing to invariant controls

• Functional redundancy analysis:

  • Create single and combinatorial CIPK mutants (e.g., cipk14, cipk14/cipk23)

  • Compare protein expression of remaining family members using specific antibodies

  • Assess compensatory expression changes in mutant backgrounds

• Substrate specificity determination:

  • Perform parallel immunoprecipitation studies for multiple CIPKs

  • Identify common and unique interacting partners using mass spectrometry

  • Validate interactions with candidate proteins like WHY1/WHY3 using reciprocal co-IP

• Pathway integration:

  • Map connections between different CIPK pathways using phospho-specific antibodies

  • Determine points of convergence and divergence in signaling networks

  • Use phospho-proteomics to identify differential substrate preferences

This comparative approach will help elucidate the unique aspects of CIPK14 function while placing it in the broader context of the CIPK family signaling network.

What considerations are important when using CIPK14 antibodies for high-throughput proteomic studies?

For successful integration of CIPK14 antibodies in high-throughput proteomic studies:

• Antibody validation for proteomics applications:

  • Verify antibody performance in immunoprecipitation followed by mass spectrometry

  • Assess background binding using appropriate negative controls

  • Determine optimal antibody-to-bead ratios for maximal specific enrichment

• Sample preparation optimization:

  • Develop consistent protein extraction protocols compatible with downstream applications

  • Standardize tissue amounts and extraction conditions across experimental samples

  • Consider fractionation approaches to enrich for CIPK14-containing complexes

• Quantitative considerations:

  • Incorporate isotope labeling approaches (SILAC, TMT) for accurate quantification

  • Include spike-in standards for normalization between experimental batches

  • Design biological and technical replicates with appropriate statistical power

• Data analysis pipeline development:

  • Create filters to discriminate true interactions from background

  • Implement statistical approaches for significance assessment

  • Develop visualization tools for complex interaction networks

• Integration with other data types:

  • Correlate proteomic findings with transcriptomic data

  • Map identified interactions onto known signaling pathways

  • Integrate with phenotypic data from genetic studies

Previous proteomic analysis has successfully identified differentially abundant proteins in CIPK14 overexpression and WHY1/3 knockout plants, revealing only five overlapping proteins between these lines that may be closely associated with CIPK14-mediated functions of WHY proteins . This approach can be expanded to study how CIPK14 affects global proteome remodeling during immune responses and other cellular processes.

How can I design experiments to resolve contradictory findings about CIPK14 function across different research studies?

To address contradictory findings about CIPK14 function across different studies:

• Standardize experimental systems:

  • Use identical plant ecotypes and growth conditions across experiments

  • Define precise developmental stages for analysis

  • Standardize stress treatment protocols (pathogen strains, concentration, duration)

• Employ multiple detection methods:

  • Combine antibody-based detection with transcript analysis

  • Validate key findings with multiple independent antibodies

  • Incorporate mass spectrometry-based protein quantification

• Address genetic background effects:

  • Generate new mutations in standardized backgrounds

  • Create complementation lines with identical promoters and terminators

  • Use CRISPR/Cas9 to introduce precise mutations rather than T-DNA insertions

• Control for environmental variables:

  • Document complete growth conditions including light quality, temperature cycles

  • Control for circadian effects by time-matched sampling

  • Consider microbiome effects in soil-grown plants

• Systematic phenotyping approach:

  • Develop quantitative assays for immune responses

  • Use image-based phenotyping for consistent scoring

  • Apply statistical models that account for experimental variability

This comprehensive approach has been valuable in resolving seemingly contradictory findings about CIPK14's role in plant immunity, showing that it functions as a negative regulator at both transcriptional and post-transcriptional levels . By implementing standardized experimental designs and rigorous controls, researchers can build a more consistent understanding of CIPK14 function across different biological contexts.