CIPK18 Antibody

What is CIPK18 and why is it significant in plant research?

CIPK18 is a member of the CBL-interacting protein kinase family, which forms part of plant-specific Ca²⁺ receptor complexes. These sensor-responder complexes comprise calcineurin B-like (CBL) proteins and CBL-interacting protein kinases (CIPKs). The CBL-CIPK module is widely involved in plant growth, development, and numerous abiotic stress response signaling pathways . CIPK18 has been specifically implicated in drought tolerance in potato (StCIPK18) and ammonium toxicity response in rice (OsCIPK18), making it a significant target for studying plant stress adaptation mechanisms .

What species-specific CIPK18 antibodies are available for research?

Several species-specific CIPK18 antibodies have been developed for research purposes. The most well-characterized is for Oryza sativa subsp. japonica (Rice) with the Uniprot number Q5W736 . Other CIPK antibodies are also available for research, including those for CIPK13 and CIPK3 in rice. When selecting an antibody, researchers should verify species cross-reactivity, especially if working with non-model plants where specific antibodies may not be commercially available.

How does CIPK18 function within the calcium signaling network in plants?

CIPK18 functions as part of the CBL-CIPK module, which acts as a calcium-sensing and signaling mechanism in plants. Upon calcium binding to CBL proteins, the CBL-CIPK complex forms, activating CIPK18's kinase activity. In studies with potato StCIPK18, it was demonstrated that this protein interacts with several CBL proteins (StCBL1, StCBL4, StCBL6, and StCBL8), with the StCIPK18-StCBL4 interaction being particularly well-verified through bimolecular fluorescence complementation (BiFC) . This interaction network enables plants to translate calcium signals into appropriate physiological responses to environmental stresses.

What are the optimal protocols for using CIPK18 antibody in Western blot analyses?

For Western blot analysis using CIPK18 antibody, researchers should follow these methodological steps:

• Extract total protein from plant tissue using a standard protein extraction buffer containing protease inhibitors

• Quantify protein concentration using Bradford or BCA assay

• Separate 20-50 μg of protein by SDS-PAGE (10-12% gel recommended)

• Transfer proteins to PVDF membrane (0.45 μm)

• Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with primary CIPK18 antibody (1:1000-1:2000 dilution) overnight at 4°C

• Wash 3× with TBST

• Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour

• Wash 3× with TBST

• Develop using ECL substrate and image

When analyzing results, researchers should note that CIPK18 protein is approximately 52-55 kDa in size, though this may vary slightly between species.

How can CIPK18 antibody be used for protein localization studies?

CIPK18 antibody can be effectively used for immunolocalization studies to determine the subcellular distribution of CIPK18. Research has shown that StCIPK18 is localized in both the cell membrane and cytoplasm . For immunolocalization:

• Fix plant tissue samples in 4% paraformaldehyde

• Prepare tissue sections (10-20 μm)

• Permeabilize with 0.1% Triton X-100

• Block with 3% BSA in PBS for 1 hour

• Incubate with CIPK18 primary antibody (1:200) overnight at 4°C

• Wash 3× with PBS

• Incubate with fluorescent-conjugated secondary antibody (1:500) for 2 hours

• Counterstain nuclei with DAPI

• Mount and visualize using confocal microscopy

For verification, parallel experiments using GFP-tagged CIPK18 expression can be conducted, which has confirmed the membrane and cytoplasmic localization pattern in previous studies .

What are the most effective immunoprecipitation conditions for studying CIPK18 protein interactions?

For immunoprecipitation to study CIPK18 protein interactions:

• Extract total protein from 5-10 g of plant tissue using a non-denaturing lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease inhibitors)

• Pre-clear lysate with protein A/G beads for 1 hour at 4°C

• Incubate pre-cleared lysate with CIPK18 antibody (5-10 μg) overnight at 4°C with gentle rotation

• Add protein A/G beads and incubate for 2-3 hours

• Wash beads 4× with wash buffer (lysis buffer with reduced detergent)

• Elute proteins with SDS sample buffer

• Analyze by Western blot or mass spectrometry

This approach has successfully identified interactions between StCIPK18 and various StCBL proteins, particularly StCBL4, which was further verified using bimolecular fluorescence complementation (BiFC) .

How does CIPK18 contribute to drought stress tolerance mechanisms in plants?

Research with potato StCIPK18 has revealed its significant role in drought stress tolerance. When StCIPK18 is overexpressed, plants show:

• Decreased water loss rate

• Increased relative water content (RWC)

• Lower malondialdehyde (MDA) content (indicating reduced lipid peroxidation)

• Increased proline accumulation (an osmoprotectant)

• Enhanced antioxidant enzyme activities (CAT, SOD, and POD)

Conversely, StCIPK18 knockout plants show the opposite effects, with increased sensitivity to drought stress. These findings suggest that CIPK18 positively regulates drought tolerance through multiple physiological mechanisms, including improved water retention, osmoregulation, and enhanced reactive oxygen species (ROS) scavenging capacity .

What is the role of CIPK18 in ammonium toxicity response in rice?

OsCIPK18 in rice plays a crucial role in the plant's response to ammonium toxicity. Studies using the T-DNA insertion mutant (cipk18) have shown that:

• The cipk18 mutant exhibits decreased sensitivity to ammonium toxicity

• Root biomass and length of cipk18 are less inhibited by excess ammonium compared to wild-type

• OsCIPK18 affects ammonium uptake by regulating the expression of transporters, particularly OsAMT1;2

• Transcriptome analysis revealed that OsCIPK18 does not affect ammonium assimilation pathways (GS/GOGAT enzyme activities remain unchanged)

• OsCIPK18 functions as a transmitter in auxin and abscisic acid (ABA) signaling pathways affected by excess ammonium

These findings indicate that OsCIPK18 is part of a regulatory network that modulates rice response to ammonium toxicity, primarily through controlling ammonium uptake rather than assimilation.

What transcriptional networks are regulated by CIPK18 under stress conditions?

Transcriptome analysis of rice under ammonium stress has identified an OsCIPK18-regulated/dependent transcriptomic network. Key components include:

• Ion transporters: particularly ammonium transporters like OsAMT1;2

• Metabolic enzymes: involved in various cellular processes

• Cell wall formation factors: affecting root architecture and development

• Phytohormone signaling components: primarily in auxin and ABA pathways

The network includes several core transcription factors that act downstream of OsCIPK18 during stress response. This transcriptional regulatory network reveals that CIPK18 has a fundamental role in coordinating various cellular responses to environmental stresses, suggesting it as a master regulator in plant stress adaptation.

How can researchers distinguish between CIPK18 and other closely related CIPK proteins in experimental settings?

Distinguishing between closely related CIPK proteins requires careful experimental design:

• Antibody Specificity: Use carefully validated antibodies for CIPK18 that have been tested against other CIPK family members. The commercially available CIPK18 antibody (CSB-PA730149XA01OFG) for rice (Q5W736) has been optimized for specificity .

• Peptide Competition Assay: Perform a peptide competition assay using synthetic peptides corresponding to unique regions of CIPK18 to confirm antibody specificity.

• 2D Gel Electrophoresis: Combine with Western blotting for better separation of closely related proteins.

• Mass Spectrometry: Identify CIPK18-specific peptides following immunoprecipitation to confirm identity.

• Expression Analysis: Combine protein analysis with gene expression studies using CIPK18-specific primers in qRT-PCR.

The high sequence similarity between CIPK family members necessitates these additional validation steps to ensure experimental results specifically reflect CIPK18 rather than related proteins.

What are the optimal experimental designs for studying CIPK18 phosphorylation targets?

To identify and validate CIPK18 phosphorylation targets:

• In vitro Kinase Assay:

  • Express and purify recombinant CIPK18

  • Incubate with potential substrates in kinase buffer containing [γ-³²P]ATP

  • Analyze phosphorylation by autoradiography or phospho-specific antibodies

• Phosphoproteomic Analysis:

  • Compare phosphoproteomes of wild-type and CIPK18 overexpression/knockout plants

  • Use SILAC or TMT labeling for quantitative analysis

  • Validate candidates by targeted MS/MS

• Yeast Two-Hybrid Combined with Mutagenesis:

  • Use CIPK18 as bait to identify interacting proteins

  • Confirm direct phosphorylation using in vitro assays

  • Create phospho-null and phospho-mimetic mutants of targets to validate functional significance

• Bimolecular Fluorescence Complementation (BiFC):

  • Verify in vivo interactions between CIPK18 and potential targets

  • This method has successfully confirmed the interaction between StCIPK18 and StCBL4

This multi-faceted approach allows for comprehensive identification and validation of genuine CIPK18 phosphorylation targets in plant stress response pathways.

How can researchers effectively use CIPK18 antibodies in chromatin immunoprecipitation (ChIP) experiments?

While CIPK18 is not a transcription factor, researchers studying its potential nuclear functions or interactions with chromatin-associated proteins may use ChIP with the following considerations:

• Crosslinking Optimization:

  • Use dual crosslinking (1% formaldehyde followed by ethylene glycol bis(succinimidyl succinate))

  • Crosslink time should be optimized (typically 10-15 minutes)

• Sonication Parameters:

  • Optimize sonication conditions to generate 200-500 bp DNA fragments

  • Verify fragment size by agarose gel electrophoresis

• Antibody Validation:

  • Confirm CIPK18 antibody specificity with Western blot prior to ChIP

  • Include IgG negative control and a positive control antibody (e.g., histone H3)

• Sequential ChIP:

  • Consider sequential ChIP if studying CIPK18 in complex with DNA-binding proteins

  • First IP with CIPK18 antibody followed by IP with antibody against the interacting transcription factor

• Data Analysis:

  • Use appropriate normalization methods (input, IgG control)

  • Validate findings with independent biological replicates

Since CIPK18 likely affects transcription indirectly through phosphorylation of transcription factors or chromatin modifiers, researchers should interpret ChIP results carefully and consider complementary approaches like ChIP-reChIP.

What are common issues when using CIPK18 antibodies and how can they be resolved?

When working with CIPK18 antibodies, researchers may encounter several challenges:

Additional considerations include species-specific optimization, as CIPK18 antibodies are often raised against specific species like rice (Oryza sativa) , and may require validation when used with other plant species.

How can researchers optimize CIPK18 protein extraction from different plant tissues?

Optimizing CIPK18 protein extraction requires tissue-specific considerations:

• Leaf Tissue:

  • Grind in liquid nitrogen to fine powder

  • Extract with buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 5 mM DTT, 0.5% Triton X-100, and protease inhibitors

  • Centrifuge at 12,000g for 15 minutes at 4°C

• Root Tissue:

  • Add PVPP (2% w/v) to extraction buffer to remove phenolics

  • Include higher concentration of protease inhibitors

  • Consider additional washing steps to remove soil contaminants

• Seed/Reproductive Tissues:

  • Use stronger extraction buffer with 2% SDS

  • Extended grinding may be necessary due to tissue hardness

  • Consider TCA/acetone precipitation to concentrate proteins

• For Membrane-Associated CIPK18:

  • Use two-phase extraction with Triton X-114

  • Alternative: microsomal fractionation followed by extraction with 1% NP-40

For all tissues, maintaining low temperature throughout extraction is critical. When analyzing results, researchers should note that CIPK18 expression levels vary significantly between tissues and developmental stages, with higher expression often observed under stress conditions .

What controls should be included when using CIPK18 antibodies in various experimental systems?

Proper experimental controls are essential when working with CIPK18 antibodies:

• Western Blot Controls:

  • Positive control: Extract from tissues known to express CIPK18 (e.g., stressed roots)

  • Negative control: Extract from CIPK18 knockout or knockdown plants

  • Loading control: Antibody against housekeeping protein (e.g., actin, tubulin)

  • Specificity control: Pre-immune serum or IgG at the same concentration

• Immunolocalization Controls:

  • Omit primary antibody control

  • Peptide competition control

  • GFP-tagged CIPK18 expression for localization verification

  • Counter-staining with organelle-specific markers

• Immunoprecipitation Controls:

  • Input sample (pre-IP lysate)

  • IgG or pre-immune serum IP

  • Unrelated antibody of same isotype

  • Reverse IP with antibodies against known interacting partners (e.g., StCBL4)

• Transgenic Expression Controls:

  • Empty vector transformants

  • Wild-type samples

  • Plants expressing unrelated protein with same tag

  • Multiple independent transgenic lines

These controls help distinguish specific CIPK18 signals from background and ensure reliable experimental interpretations when studying CIPK18's role in stress response mechanisms.

How can researchers effectively analyze CIPK18 phosphorylation state changes during stress responses?

Analyzing CIPK18 phosphorylation dynamics requires specialized approaches:

• Phospho-specific Antibodies:

  • Use antibodies that specifically recognize phosphorylated residues of CIPK18

  • Compare ratio of phosphorylated to total CIPK18 across conditions

• Phos-tag™ SDS-PAGE:

  • Incorporate Phos-tag™ acrylamide into gels to separate phosphorylated from non-phosphorylated forms

  • Visualize with standard CIPK18 antibody

  • Quantify band shifting patterns to assess phosphorylation levels

• Mass Spectrometry-Based Approaches:

  • Immunoprecipitate CIPK18 followed by tryptic digestion

  • Enrich phosphopeptides using TiO₂ or IMAC

  • Perform LC-MS/MS to identify specific phosphorylation sites

  • Use label-free quantification or isotopic labeling to compare phosphorylation levels between conditions

• Time-Course Analysis:

  • Sample at multiple timepoints after stress application

  • Create phosphorylation profiles to understand signaling dynamics

  • Correlate with downstream physiological responses

These approaches can reveal how environmental stresses like drought or ammonium toxicity influence CIPK18 activity through post-translational modifications, providing insight into activation mechanisms of plant stress signaling pathways.

What approaches can be used to study the relationship between CIPK18 and other stress-related signaling components?

To investigate CIPK18's position within stress signaling networks:

• Epistasis Analysis:

  • Generate double mutants between cipk18 and other stress signaling components

  • Compare phenotypes under stress conditions

  • Determine genetic hierarchies based on dominant phenotypes

• Co-immunoprecipitation Coupled with Mass Spectrometry:

  • Use CIPK18 antibody for immunoprecipitation from stressed and non-stressed tissues

  • Identify interacting proteins by mass spectrometry

  • Verify interactions of interest with reciprocal co-IP or BiFC

• Phosphoproteomics:

  • Compare phosphoproteomes of wild-type and cipk18 mutants

  • Identify differentially phosphorylated proteins as potential CIPK18 targets

  • Construct signaling networks based on phosphorylation patterns

• Transcriptome Analysis:

  • Compare transcriptional responses to stress in wild-type and cipk18 plants

  • Research has identified transcriptional networks regulated by OsCIPK18 under ammonium stress

  • Use bioinformatics to identify enriched cis-regulatory elements in co-regulated genes

• Hormone Signaling Integration:

  • Monitor hormone levels in wild-type vs. cipk18 plants under stress

  • Studies suggest OsCIPK18 functions in auxin and ABA signaling pathways

  • Apply exogenous hormones to test for rescue of mutant phenotypes

These multi-faceted approaches provide complementary data to position CIPK18 within the broader context of plant stress response networks.

How can CIPK18 antibody-based research be integrated with other 'omics approaches for comprehensive stress response studies?

Integrating CIPK18 antibody research with multi-omics approaches enables comprehensive understanding of stress response mechanisms:

• Integration with Transcriptomics:

  • Combine ChIP-seq (using antibodies against transcription factors identified as CIPK18 interactors) with RNA-seq

  • Correlate CIPK18 phosphorylation state with gene expression patterns

  • Identify transcription factors whose activity is modulated by CIPK18

  • Example: OsCIPK18 regulates ammonium toxicity response through a transcriptomic network

• Integration with Metabolomics:

  • Compare metabolite profiles of wild-type and cipk18 plants under stress

  • Focus on stress-related metabolites (e.g., proline in drought stress)

  • Trace metabolic pathways affected by CIPK18 activity

• Integration with Phenomics:

  • Perform high-throughput phenotyping of CIPK18 variant lines

  • Correlate CIPK18 expression/activity (detected via antibodies) with phenotypic traits

  • Example: StCIPK18 overexpression reduces water loss rate and increases relative water content under drought

• Integration with Interactomics:

  • Use protein microarrays with recombinant CIPK18 to identify interactors at scale

  • Validate hits with traditional antibody-based approaches (co-IP, BiFC)

  • Known interactions include StCIPK18 with StCBL1, StCBL4, StCBL6, and StCBL8

• Network Biology Approaches:

  • Construct integrated networks incorporating protein-protein interactions, phosphorylation events, and transcriptional regulation

  • Identify feedback loops and regulatory hubs

  • Model information flow through CIPK18-dependent pathways during stress response

This integrative approach provides systems-level insights into CIPK18 function that would not be apparent from any single methodology, advancing our understanding of plant stress adaptation mechanisms.

What emerging technologies might enhance CIPK18 antibody-based research in the future?

Several cutting-edge technologies show promise for advancing CIPK18 research:

• Proximity Labeling Techniques:

  • BioID or TurboID fusions with CIPK18 to identify proximal proteins in living cells

  • APEX2-based proximity labeling for temporal mapping of CIPK18 interactome

  • These approaches could reveal transient interactions missed by traditional co-IP

• Single-Cell Proteomics:

  • Analysis of CIPK18 levels and modifications at single-cell resolution

  • Reveal cell-type specific roles in heterogeneous tissues

  • Understand cellular heterogeneity in stress response

• Super-Resolution Microscopy:

  • Nanoscale visualization of CIPK18 localization using antibodies with fluorescent tags

  • Study dynamic changes in CIPK18 distribution during stress response

  • Resolve subcellular compartmentalization beyond traditional confocal microscopy

• CRISPR-Based Protein Tagging:

  • Endogenous tagging of CIPK18 for live-cell imaging

  • Generation of CIPK18 variants to study structure-function relationships

  • Precise mutagenesis of interaction domains identified through antibody-based studies

• Antibody Engineering:

  • Development of single-domain antibodies (nanobodies) against CIPK18

  • Creation of intrabodies for tracking CIPK18 in living cells

  • Phospho-specific antibodies for tracking CIPK18 activation status

These technologies would complement traditional antibody applications while addressing current limitations in studying dynamic processes and rare cell populations in plant stress response research.

How should researchers design experiments to study CIPK18 function across multiple plant species?

Designing cross-species studies of CIPK18 requires careful consideration:

• Sequence and Structure Analysis:

  • Perform phylogenetic analysis of CIPK18 across species of interest

  • Identify conserved domains and species-specific variations

  • Design antibodies targeting highly conserved epitopes for cross-species applications

• Antibody Validation Strategy:

  • Test commercial antibodies like the rice CIPK18 antibody (CSB-PA730149XA01OFG) against proteins from multiple species

  • Consider developing new antibodies against conserved peptides

  • Include positive controls from species with confirmed reactivity

• Comparative Expression Analysis:

  • Study CIPK18 expression patterns across species under standardized stress conditions

  • Compare subcellular localization using immunofluorescence

  • Quantify expression levels using calibrated Western blotting

• Functional Complementation:

  • Express CIPK18 orthologues from different species in cipk18 mutant background

  • Assess rescue of phenotypes such as drought sensitivity or ammonium toxicity response

  • Identify conserved and divergent functional aspects

• Interaction Conservation:

  • Test whether CIPK18-CBL interactions (like those documented between StCIPK18 and StCBL proteins) are conserved across species

  • Use yeast two-hybrid or BiFC with proteins from multiple species

This approach allows researchers to distinguish between core conserved functions of CIPK18 and species-specific adaptations, providing evolutionary insights into plant stress response mechanisms.

What are the potential applications of CIPK18 research for improving crop stress tolerance?

CIPK18 research has several promising applications for agricultural improvement:

• Marker-Assisted Selection:

  • Develop molecular markers based on favorable CIPK18 alleles

  • Select for variants associated with enhanced stress tolerance

  • Screen germplasm collections for natural variation in CIPK18 sequence and expression

• Genetic Engineering Approaches:

  • Overexpress CIPK18 to enhance drought tolerance, as demonstrated in potato

  • Modify CIPK18 expression to improve ammonium utilization and reduce toxicity in rice

  • Fine-tune expression using stress-inducible or tissue-specific promoters

• CRISPR-Based Genome Editing:

  • Introduce beneficial CIPK18 alleles from wild relatives into elite crop varieties

  • Modify regulatory regions to optimize expression patterns

  • Target interacting partners identified through antibody-based research

• Screening Technologies:

  • Develop high-throughput assays using CIPK18 antibodies to screen for compounds that modulate its activity

  • Identify chemical priming agents that enhance stress tolerance via CIPK18 pathways

• Precision Agriculture Applications:

  • Develop biosensors using CIPK18 antibodies to monitor plant stress status

  • Use CIPK18 expression/phosphorylation as biomarkers for early stress detection

  • Optimize resource application based on CIPK18-related stress indicators

These applications could lead to crops with improved tolerance to drought and nitrogen-related stresses, addressing major challenges in sustainable agriculture under changing climatic conditions.