CIPK11 Antibody

What is CIPK11 and why is it important in plant research?

CIPK11 is a serine/threonine protein kinase that forms part of the CIPK gene family, which interacts with calcineurin B-like (CBL) calcium sensors in plants. It plays significant roles in multiple signaling pathways and stress responses. CIPK11 functions as a negative regulator in drought stress response, as demonstrated by studies showing that CIPK11 overexpression (CIPK11OE) plants exhibit hypersensitivity to drought conditions through increased water loss and enhanced reactive oxygen species (ROS) accumulation . Importantly, CIPK11 interacts with and phosphorylates the transcription factor Di19-3, establishing its role in the drought stress signaling cascade . CIPK11 is also involved in programmed cell death (PCD) during plant immune responses, making it a valuable target for researchers studying plant stress biology and disease resistance mechanisms .

What is the cellular localization of CIPK11 and why is this relevant for antibody-based detection?

CIPK11 exhibits dual localization in plant cells. Fluorescent labeling studies reveal that CIPK11, like several other CIPK family members (CIPK1, CIPK2, CIPK3, CIPK4, CIPK7, CIPK8, CIPK10, CIPK14, CIPK17, CIPK21, CIPK23, and CIPK24), shows significant fluorescence in both the cytoplasm and nucleus . Subsequent studies have confirmed this localization pattern, with specific research demonstrating that CIPK11 localizes in the cytoplasm and nucleus in epidermal cells of tobacco leaves . This dual localization is critical for antibody-based detection experiments because:

• Different fixation protocols may be required to preserve protein integrity in different cellular compartments

• Background signals may vary between compartments, requiring optimization of antibody dilutions

• Co-localization studies should account for the protein's distribution across multiple compartments

• Nuclear localization may require additional permeabilization steps for optimal antibody penetration

How do CIPK11 interactions with binding partners influence antibody selection?

CIPK11 interacts with multiple proteins that may affect epitope accessibility for antibodies. Key interactions include:

• CBL proteins: CIPK11 forms functional complexes with specific CBL calcium sensors. In tomato, studies show that Cbl10 interacts specifically with Cipk6 but not with Cipk11 or Cipk14, suggesting selective binding among family members .

• Transcription factor Di19-3: CIPK11 directly interacts with Di19-3, a Cys2/His2-type zinc-finger transcription factor involved in drought stress response. This interaction occurs specifically in the nucleus, as confirmed by bimolecular fluorescence complementation (BiFC) assays .

• RbohB: Evidence suggests that Cbl10 and Cipk6 interact with RbohB at the plasma membrane, establishing a potential link between calcium signaling and ROS generation .

When selecting antibodies for CIPK11 detection, researchers should consider these interactions, as antibodies targeting regions involved in protein-protein binding may show reduced accessibility in interaction complexes. Epitope mapping relative to known interaction domains can help select antibodies that remain effective regardless of CIPK11's interaction status.

How can CIPK11 antibodies be optimized for detecting phosphorylation states?

CIPK11 undergoes autophosphorylation and phosphorylates downstream targets, making phosphorylation state-specific antibodies valuable research tools. Based on experimental evidence, CIPK11 demonstrates clear autophosphorylation activity, as evidenced by a ~60 kDa band detected using an anti-phosphothreonine antibody in kinase activity assays . To optimize antibodies for detecting CIPK11 phosphorylation states:

• Use phospho-specific antibodies targeting known autophosphorylation sites on CIPK11

• Employ lambda phosphatase treatments as negative controls to confirm phospho-specific signals

• Consider developing custom phospho-specific antibodies against verified phosphorylation sites in CIPK11

• Use kinase-dead CIPK11 mutants as negative controls in phosphorylation assays

• Implement comparative immunoblotting with general CIPK11 antibodies to assess phosphorylation ratios

For validation, researchers should confirm specificity using CIPK11 knockout/knockdown plant materials, such as the cipk11 mutant line (Salk_108074) which has a T-DNA insertion 456 bp downstream of the predicted ATG start site and shows complete disruption of CIPK11 expression .

What experimental designs are most effective for studying CIPK11's role in drought stress using antibody-based methods?

Based on CIPK11's established role as a negative regulator in drought stress, the following experimental approaches using antibody-based methods are recommended:

• Time-course immunoblotting analysis:

  • Monitor CIPK11 protein levels during progressive drought stress (0-8 days)

  • Compare wild-type, cipk11 mutant, and CIPK11OE plants

  • Correlate protein levels with physiological measurements of drought stress

• Co-immunoprecipitation for stress-specific interactions:

  • Use anti-CIPK11 antibodies to immunoprecipitate protein complexes from drought-stressed and control plants

  • Identify differential interaction partners under stress conditions

  • Confirm specific interactions with Di19-3 and other drought-response factors

• Chromatin immunoprecipitation (ChIP) for transcriptional regulation:

  • Perform ChIP experiments using antibodies against Di19-3 in wild-type vs. cipk11 mutants

  • Analyze binding to promoters of drought-responsive genes (RAB18, RD29A, RD29B, DREB2A)

  • Correlate with CIPK11-dependent phosphorylation status of Di19-3

• Immunohistochemistry for tissue-specific localization:

  • Compare CIPK11 localization patterns in drought-resistant and sensitive tissues

  • Assess nuclear translocation rates under stress conditions

  • Quantify co-localization with known drought response factors

These approaches should incorporate appropriate controls including the cipk11 mutant, which shows slight drought tolerance compared to wild-type, and CIPK11OE plants that exhibit extreme drought sensitivity with survival rates of only 15-18% compared to 82% in wild-type plants after drought treatment .

What are the challenges in detecting endogenous CIPK11 versus overexpressed CIPK11?

Detecting endogenous versus overexpressed CIPK11 presents several technical challenges for antibody-based research:

For endogenous detection, researchers should incorporate appropriate positive controls such as tissue types with known higher CIPK11 expression and consider concentration steps such as immunoprecipitation before detection. For overexpressed CIPK11, epitope tags (such as Flag used in CIPK11OE plants) can facilitate detection using tag-specific antibodies alongside CIPK11-specific antibodies for confirmation.

What are the optimal sample preparation protocols for CIPK11 immunodetection in different plant tissues?

Based on experimental evidence and CIPK11's dual localization, the following sample preparation protocols are recommended:

• For total protein extraction (immunoblotting):

  • Extract tissues in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100

  • Include protease inhibitors (PMSF, leupeptin, aprotinin)

  • Add phosphatase inhibitors (sodium fluoride, sodium orthovanadate) to preserve phosphorylation

  • Use reducing agents (DTT or β-mercaptoethanol) to maintain protein solubility

  • For drought stress experiments, normalize loading by tissue fresh weight rather than protein concentration

• For nuclear/cytoplasmic fractionation:

  • Implement gentle cell lysis to maintain nuclear integrity

  • Use sucrose gradient-based separation for clean nuclear fractions

  • Verify fraction purity with compartment-specific markers

  • Optimize salt concentration to extract nuclear CIPK11 without disrupting DNA-protein interactions

• For immunoprecipitation:

  • Use mild detergents (0.1% NP-40) to preserve protein-protein interactions

  • Implement stepwise extraction to compare easily soluble versus tightly bound fractions

  • Consider crosslinking for transient interactions

  • Compare native versus denaturing conditions to distinguish direct versus indirect interactions

These protocols should be validated using appropriate controls including the cipk11 mutant line and CIPK11OE plants that express CIPK11 at levels 43-52 times higher than wild-type .

What controls are essential for validating CIPK11 antibody specificity?

To ensure robust and reproducible results with CIPK11 antibodies, the following controls are essential:

• Genetic controls:

  • cipk11 knockout/knockdown lines (e.g., Salk_108074 T-DNA insertion line)

  • CIPK11 overexpression lines (e.g., CIPK11OE#1 and CIPK11OE#8)

  • Plants with tagged versions of CIPK11 (e.g., Flag-tagged CIPK11) for dual detection

• Biochemical controls:

  • Pre-absorption of antibody with purified recombinant CIPK11 protein

  • Competition assays with related CIPK family members (particularly those with highest sequence homology)

  • Immunodepleted samples to confirm signal specificity

  • Gradient dilution series to confirm signal linearity

• Experimental validation:

  • Multiple antibodies targeting different epitopes of CIPK11

  • Correlation between protein detection and known transcript levels

  • Mass spectrometry confirmation of immunoprecipitated proteins

  • siRNA/VIGS-mediated silencing for partial knockdown validation

CIPK11 belongs to a family with multiple members sharing sequence similarity, so cross-reactivity testing is particularly important. For example, in tomato, Cipk6 shows high amino acid similarity to Cipk11 and Cipk14 , suggesting potential cross-reactivity that must be controlled for.

How can immunoprecipitation be optimized for studying CIPK11 interactions with CBLs and transcription factors?

Optimizing immunoprecipitation (IP) protocols for CIPK11 interaction studies requires careful consideration of the nature of these interactions. Based on experimental evidence:

• Buffer composition considerations:

  • Include Ca²⁺ (1-2 mM) in buffers to stabilize CBL-CIPK interactions, as these are calcium-dependent

  • Use phosphatase inhibitors to preserve phosphorylation-dependent interactions

  • Test different salt concentrations (150-300 mM) to optimize specificity versus recovery

  • Include mild detergents (0.1% NP-40 or 0.5% Triton X-100) to solubilize membrane-associated complexes

• Targeting strategy options:

  • Direct IP using anti-CIPK11 antibodies

  • Reverse IP using antibodies against known interaction partners (CBLs, Di19-3)

  • Tandem IP for higher specificity (sequential purification using two different antibodies)

  • Tag-based IP if using epitope-tagged constructs (as in CIPK11OE-Flag plants)

• Validation approaches:

  • Reciprocal co-IP (IP with anti-CIPK11 and detect partner, then IP with anti-partner and detect CIPK11)

  • Comparison with yeast two-hybrid and BiFC results for known interactions

  • Use truncated protein variants to map interaction domains

  • Include interaction-deficient mutants as negative controls

The experimental design should account for interaction specificity. For example, studies have demonstrated that Cbl10 did not interact with Cipk11 or Cipk14 (tomato CIPK proteins with high amino acid similarity to Cipk6), suggesting that the Cipk6/Cbl10 interaction is specific . This shows that different CIPKs have distinct CBL interaction profiles that must be considered when interpreting co-IP results.

How to overcome common challenges in CIPK11 immunodetection experiments?

CIPK11 immunodetection can present several challenges based on its properties and expression patterns. Here are solutions for common issues:

• Weak signal from endogenous CIPK11:

  • Use enhanced chemiluminescence detection systems

  • Implement sample concentration techniques (TCA precipitation, methanol/chloroform precipitation)

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

  • Use signal amplification systems (biotin-streptavidin)

  • Consider tissue selection based on known expression patterns

• High background in immunolocalization:

  • Optimize blocking with 5% BSA with 0.1% Triton X-100

  • Include competing proteins (non-fat dry milk) in antibody dilution

  • Increase washing steps (use at least 3 × 10-minute washes)

  • Pre-absorb secondary antibodies with plant tissue powder

  • Use CIPK11 knockout tissues as negative controls

• Non-specific bands in immunoblotting:

  • Validate with recombinant CIPK11 as a migration standard

  • Compare wild-type and cipk11 mutant samples side by side

  • Use gradient gels to improve separation of similar molecular weight proteins

  • Implement more stringent washing conditions

  • Consider using monoclonal antibodies for higher specificity

• Variability between experiments:

  • Standardize tissue harvesting time (CIPK11 may have diurnal expression patterns)

  • Consider stress conditions that affect CIPK11 expression (e.g., drought stress upregulates CIPK11 transcripts 3.0-3.24 fold)

  • Establish positive control samples that can be included in each experiment

  • Use internal loading controls appropriate for the experimental conditions

What techniques are recommended for studying CIPK11 phosphorylation activity in vitro?

CIPK11 shows autophosphorylation and can phosphorylate targets like Di19-3 . The following techniques are recommended for studying its phosphorylation activity:

• In vitro kinase assays:

  • Express and purify recombinant CIPK11 using bacterial expression systems (e.g., GST-CIPK11-KD)

  • Include purified substrates (e.g., GST-Di19-3)

  • Use [γ-³²P]ATP for radioactive detection or phospho-specific antibodies

  • Include appropriate controls (kinase-dead CIPK11, known substrates)

  • Test dependency on Ca²⁺ and CBL proteins

• Antibody-based detection methods:

  • Use anti-phosphothreonine antibodies to detect autophosphorylation

  • Develop or obtain phospho-specific antibodies for key autophosphorylation sites

  • Implement Western blotting following kinase reactions

  • Use Phos-tag™ SDS-PAGE to enhance separation of phosphorylated from non-phosphorylated proteins

• Quantitative analysis approaches:

  • Implement time-course assays to determine kinetics

  • Use varying substrate concentrations to determine Km values

  • Compare wild-type versus mutant CIPK11 activities

  • Assess effects of regulatory factors (CBLs, calcium, pH, redox state)

A sample protocol based on published work would include:

• Express GST-CIPK11-KD and potential substrates in E. coli

• Purify using glutathione S-Sepharose 4B resin

• Incubate purified proteins in kinase buffer containing ATP

• Separate proteins by SDS-PAGE

• Detect phosphorylated proteins using anti-phosphothreonine antibody

• Visualize total proteins with Coomassie Brilliant Blue (CBB) staining

How can antibody-based methods help elucidate CIPK11's role in calcium signaling pathways?

CIPK11 is part of the CBL-CIPK calcium sensing and signaling pathway in plants. Antibody-based methods can help elucidate several aspects of this role:

• Calcium-dependent complex formation:

  • Use co-immunoprecipitation with anti-CIPK11 antibodies in the presence/absence of calcium

  • Implement in situ proximity ligation assays to visualize CIPK11-CBL interactions in intact cells

  • Compare complex formation under different calcium concentrations

  • Identify changes in interaction partners under varying calcium conditions

• Subcellular translocation in response to calcium signals:

  • Perform immunofluorescence microscopy under calcium-inducing conditions

  • Quantify nuclear/cytoplasmic ratios of CIPK11 following calcium flux

  • Use cell fractionation followed by immunoblotting to track CIPK11 movement

  • Compare wild-type CIPK11 with calcium-binding deficient mutants

• Calcium-dependent phosphorylation cascades:

  • Map phosphorylation events downstream of CIPK11 activation using phospho-specific antibodies

  • Compare phosphorylation patterns in the presence of calcium chelators

  • Identify calcium-dependent substrates by differential phosphoproteomic analysis

  • Use phosphorylation state-specific antibodies to monitor CIPK11 activation status

• Integration with other signaling pathways:

  • Use multiplex immunolabeling to co-visualize CIPK11 with components of other pathways

  • Perform sequential immunoprecipitation to identify multi-protein complexes

  • Compare interaction networks under different calcium and stress conditions

  • Use antibody-based protein arrays to assess multiple pathway components simultaneously

These approaches could help establish CIPK11's role in the CIPK-CBL complex that raises cytosolic free Ca²⁺ levels, enhances CIPK kinase activity, and triggers phosphorylation cascades as described in the literature .

How might newly developed antibody technologies advance CIPK11 research?

Emerging antibody technologies offer exciting possibilities for advancing CIPK11 research:

• Single-domain antibodies (nanobodies):

  • Develop anti-CIPK11 nanobodies for live-cell imaging

  • Use conformation-specific nanobodies to detect active versus inactive CIPK11

  • Implement intrabodies to track CIPK11 dynamics in living plant cells

  • Apply nanobody-based proximity labeling to map the CIPK11 interactome in situ

• Bifunctional antibodies:

  • Create CIPK11-targeting degraders using antibody-PROTAC conjugates

  • Develop antibody-based CIPK11 inhibitors for acute functional studies

  • Design split-reporter systems based on bifunctional antibodies for interaction detection

  • Implement transcription factor-recruiting antibodies to control CIPK11 expression

• Spatiotemporal control technologies:

  • Apply optogenetic control to CIPK11-antibody systems for light-controlled studies

  • Develop chemically-induced proximity systems for acute manipulation of CIPK11 localization

  • Implement rapid degradation techniques for temporal protein control

  • Use spatial patterning of immobilized antibodies to study localized CIPK11 functions

• High-throughput screening approaches:

  • Develop antibody-based sensors for CIPK11 activity in plant cell arrays

  • Implement multiplex antibody-based detection for pathway component analysis

  • Create CIPK11 substrate identification platforms using antibody-based detection

  • Use antibody-based proteomics to identify novel CIPK11 targets across different stress conditions

These technologies would help address key knowledge gaps about CIPK11, such as its role in post-transcriptional modification of plant signaling pathways and abiotic resistance , and its involvement in phosphorylation during cold stress .

What research questions about CIPK11 remain unanswered that antibody-based approaches could address?

Despite significant progress in understanding CIPK11 function, several key questions remain that could be addressed using antibody-based approaches:

• Comprehensive interactome mapping:

  • Which proteins interact with CIPK11 under different stress conditions?

  • How does the CIPK11 interactome change during plant development?

  • What is the stoichiometry of CIPK11-containing complexes?

  • How do post-translational modifications affect CIPK11 interaction networks?

• Regulation of CIPK11 activity:

  • What is the full spectrum of CIPK11 substrates across different plant tissues?

  • How is CIPK11 activity regulated beyond calcium/CBL binding?

  • What is the phosphorylation status of CIPK11 under different stress conditions?

  • How do other signaling pathways cross-talk with CIPK11-mediated responses?

• Spatial and temporal dynamics:

  • What is the subcellular distribution of CIPK11 during stress responses?

  • How rapidly does CIPK11 relocalize following calcium signals?

  • What is the half-life of CIPK11 under normal versus stress conditions?

  • How does CIPK11 contribute to local versus systemic stress responses?

• Functional conservation across species:

  • How conserved are CIPK11 epitopes across different plant species?

  • Do CIPK11 orthologs interact with the same partners across species?

  • Are there species-specific post-translational modifications of CIPK11?

  • How does CIPK11 function differ between monocots and dicots?

Antibody-based approaches would be particularly valuable for addressing these questions, especially considering that most current knowledge about CIPK11 comes from genetic studies using overexpression lines (CIPK11OE) and knockout mutants (cipk11) , which provide limited information about protein dynamics and interactions.