CIPK19 Antibody

What is CIPK19 and how does it function in plant stress response mechanisms?

CIPK19 belongs to the CIPK family of protein kinases that interact with calcium sensor CBL (Calcineurin B-Like) proteins to form signaling complexes involved in plant stress responses. Based on research on related proteins like CIPK9, CIPK19 likely functions in signal transduction pathways activated during various environmental stresses. CIPK9 has been demonstrated to interact with calcium sensors CBL2 and CBL3, playing crucial roles in K+ homeostasis under low-K+ stress conditions . Similarly, CIPK19 presumably forms protein complexes with specific CBL calcium sensors, which then target and phosphorylate downstream proteins involved in stress adaptation mechanisms.

What experimental applications are CIPK19 antibodies suitable for?

CIPK19 antibodies can be utilized in multiple research applications:

The choice of application should be guided by research objectives. For protein-protein interaction studies, immunoprecipitation methods similar to those used for CIPK9-CBL2/3 interactions would be appropriate .

How can I determine if my CIPK19 antibody has adequate specificity?

Ensuring antibody specificity is critical for reliable research outcomes:

• Cross-reactivity testing: Test against recombinant proteins from multiple CIPK family members to assess potential cross-reactivity.

• Knockout validation: Verify absence of signal in CIPK19 knockout/knockdown plant tissues.

• Peptide competition: Pre-incubation with the immunizing peptide should abolish specific signal.

• Western blot analysis: A single band at the expected molecular weight indicates good specificity.

• Mass spectrometry validation: Confirm identity of immunoprecipitated proteins.

For CBL-CIPK interactions, approaches similar to those used for CIPK9 can be applied, where pull-down assays with GST-tagged CBL proteins followed by immunoblotting confirmed the interaction specificity .

What are the optimal conditions for using CIPK19 antibodies in Western blot analyses?

For successful Western blot detection of CIPK19:

• Sample preparation: Extract total proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, protease inhibitors, and phosphatase inhibitors.

• Protein separation: Load 20-40 μg total protein on 10-12% SDS-PAGE gels.

• Transfer conditions: Transfer to PVDF membrane at 100V for 1 hour or 30V overnight.

• Blocking: Use 5% non-fat milk in TBST for 1 hour at room temperature.

• Antibody incubation: Dilute primary CIPK19 antibody 1:1000-1:2000 in 5% BSA/TBST and incubate overnight at 4°C.

• Detection: After secondary antibody incubation and washing, visualize using ECL substrate.

• Controls: Include positive control (tissue known to express CIPK19) and negative control (pre-immune serum).

This approach aligns with successful detection methods used for related proteins like CIPK9, where cMyc-CIPK9 fusion proteins were detected through immunoblotting after pull-down assays .

How can I optimize co-immunoprecipitation protocols for studying CIPK19 interactions with calcium sensors?

For co-immunoprecipitation of CIPK19 and its interaction partners:

• Buffer optimization: Use mild lysis buffers (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, protease inhibitors) to preserve protein-protein interactions.

• Pre-clearing: Incubate lysates with Protein A/G beads before immunoprecipitation to reduce non-specific binding.

• Antibody binding: Incubate 2-5 μg antibody per mg protein overnight at 4°C with gentle rotation.

• Reciprocal IP: Perform reverse co-IP using antibodies against suspected binding partners.

• Calcium dependency: Include calcium (1-2 mM) or EGTA in buffers to test calcium-dependent interactions.

• Elution conditions: Use gentle elution with peptide competition or more stringent SDS elution depending on downstream applications.

Research on CIPK9 has successfully demonstrated protein interactions using GST-tagged CBL2 or CBL3 to pull down cMyc-CIPK9 fusion proteins from plant extracts, confirming their interaction in planta .

What approaches can be used to study CIPK19 phosphorylation states and kinase activity?

To investigate CIPK19 phosphorylation and activity:

• Phospho-specific antibodies: Generate antibodies against predicted phosphorylation sites on CIPK19.

• In vitro kinase assays:

  • Express recombinant CIPK19 in E. coli or insect cells

  • Test autophosphorylation in the presence of [γ-32P]ATP

  • Assess substrate phosphorylation using candidate proteins

  • Include CBL calcium sensors to evaluate activation mechanisms

• Mass spectrometry analysis:

  • Immunoprecipitate CIPK19 from plants under different stress conditions

  • Perform phosphopeptide enrichment

  • Identify phosphorylation sites by LC-MS/MS

• Phosphorylation site mutagenesis:

  • Generate phospho-null (S/T to A) and phosphomimetic (S/T to D/E) mutants

  • Assess impact on kinase activity and protein interactions

  • Evaluate phenotypic consequences in planta

This approach builds on our understanding of CIPK activation mechanisms, where interaction with calcium-bound CBL proteins triggers conformational changes and activation of the kinase domain.

How can I address cross-reactivity issues with antibodies against different CIPK family members?

Cross-reactivity is a common challenge when working with protein families like CIPKs:

• Epitope selection strategies:

  • Target unique regions of CIPK19, particularly in the C-terminal domain

  • Avoid conserved kinase domains or NAF/FISL motifs that are similar across CIPK proteins

  • Use sequence alignment to identify CIPK19-specific regions

• Antibody validation approaches:

  • Test against multiple recombinant CIPK proteins

  • Use tissues from CIPK19 knockout plants as negative controls

  • Perform peptide competition assays with specific and non-specific peptides

• Cross-reactivity mitigation:

  • Pre-absorb antibodies with recombinant proteins of closely related CIPKs

  • Optimize antibody dilutions to reduce non-specific binding

  • Increase stringency of washing steps in immunoassays

A particular challenge with CIPK proteins is their structural similarity, as they all contain kinase domains and CBL-binding motifs, requiring careful antibody design and validation.

What are common challenges in detecting CIPK19 in different plant tissues and how can they be overcome?

Detecting endogenous CIPK19 presents several challenges:

• Low expression levels: CIPK proteins often show low basal expression, similar to CIPK9 which is primarily activated under specific stress conditions like low K+ environments .

• Tissue-specific challenges:

• Stress-induced expression: Expression may increase only under specific stress conditions, requiring appropriate experimental treatments to detect CIPK19.

• Post-translational modifications: Phosphorylation status may affect antibody recognition; include phosphatase inhibitors during extraction.

• Protein degradation: Use fresh tissue and include multiple protease inhibitors in extraction buffers.

How does sample preparation affect CIPK19 detection and what modifications can improve results?

Sample preparation significantly impacts CIPK19 detection success:

• Extraction buffer optimization:

  • Include 10 mM DTT or β-mercaptoethanol to maintain reduced state

  • Add phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4) to preserve phosphorylation

  • Use protease inhibitor cocktail to prevent degradation

  • Test different detergents (Triton X-100, NP-40, CHAPS) for optimal extraction

• Subcellular fractionation:

  • Enrich for membrane fractions where CIPK-CBL complexes may localize

  • Separate nuclear and cytosolic fractions to determine subcellular distribution

  • Use density gradient centrifugation for organelle separation

• Protein precipitation methods:

  • TCA/acetone precipitation can concentrate proteins but may affect epitope recognition

  • Methanol/chloroform precipitation often preserves immunoreactivity better

• Sample storage considerations:

  • Avoid repeated freeze-thaw cycles

  • Store samples in small aliquots at -80°C

  • Add glycerol (10%) for cryoprotection

The CBL-CIPK calcium signaling system is crucial for plants to respond to environmental stimuli , and proper sample preparation is essential to maintain protein integrity for accurate analysis.

How can CIPK19 antibodies be used to investigate protein-protein interaction networks in stress signaling pathways?

CIPK19 antibodies can be powerful tools for mapping protein interaction networks:

• Co-immunoprecipitation coupled with mass spectrometry:

  • Immunoprecipitate CIPK19 from plants under different stress conditions

  • Identify co-precipitating proteins by mass spectrometry

  • Compare interactomes across different stresses to identify condition-specific interactions

• Proximity labeling approaches:

  • Generate CIPK19 fusions with BioID or APEX2

  • Identify proteins in close proximity to CIPK19 in living cells

  • Compare labeled proteins under different stress conditions

• Yeast three-hybrid validation:

  • Confirm CIPK19-CBL-target protein interactions

  • Test calcium dependency of interactions

  • Investigate competition between different interaction partners

• In situ proximity ligation assay (PLA):

  • Visualize CIPK19 interactions with specific partners in plant tissues

  • Quantify interactions under different environmental conditions

Similar approaches have been successful in identifying interacting partners for CIPK9, revealing its interaction with calcium sensors CBL2 and CBL3 in planta through pull-down assays .

What experimental designs are most effective for studying CIPK19's role in drought and stress responses?

To investigate CIPK19's role in stress responses, consider these experimental approaches:

• Genetic approaches:

  • Generate CIPK19 knockout/knockdown mutants

  • Create CIPK19 overexpression lines

  • Develop complementation lines with wild-type or mutated CIPK19

• Phenotypic analysis under stress conditions:

  • Subject plants to controlled drought, salt, cold or nutrient stress

  • Monitor physiological parameters (water loss, electrolyte leakage, ROS levels)

  • Measure stress hormone levels (ABA, ethylene)

  • Assess K+ content and homeostasis under stress conditions

• Transcriptomic and proteomic analysis:

  • Compare wild-type and CIPK19-modified plants under stress

  • Identify genes and proteins regulated by CIPK19

  • Perform temporal analysis to track signaling progression

• Phosphoproteomic screening:

  • Identify proteins differentially phosphorylated in cipk19 mutants

  • Confirm direct substrates through in vitro kinase assays

  • Validate the functional significance of phosphorylation events

Research on the related CBL1/9-CIPK1 calcium sensor system has shown its involvement in drought stress responses , suggesting similar roles for other CBL-CIPK combinations including potentially CIPK19.

How can active learning approaches improve the prediction of CIPK19-antigen binding for antibody development?

Active learning methods can enhance CIPK19 antibody development:

• Library-on-library screening approaches:

  • Test multiple antibody candidates against CIPK19 variants

  • Iteratively expand labeled datasets based on binding outcomes

  • Apply machine learning to predict optimal epitopes and binding affinities

• Out-of-distribution prediction improvements:

  • Use simulation frameworks like Absolut! to evaluate antibody performance

  • Implement machine learning algorithms that can analyze many-to-many relationships

  • Reduce required experimental testing by 28-35% compared to random screening approaches

• Strategic epitope mapping:

  • Focus on regions with high predicted antigenicity

  • Target epitopes conserved across plant species for broad applicability

  • Avoid regions prone to post-translational modifications

• Computational design optimization:

  • Use structural modeling to predict antibody-antigen interactions

  • Optimize complementarity-determining regions (CDRs)

  • Test binding against related CIPK family members in silico

Recent research has shown that active learning strategies can significantly improve experimental efficiency in antibody development, reducing the number of required experimental variants by up to 35% .

How do antibodies against CIPK19 compare with those targeting other CIPK family members?

Comparing antibodies across the CIPK family reveals important considerations:

• Epitope conservation analysis:

  • CIPK kinase domains share high sequence similarity, increasing cross-reactivity risk

  • C-terminal regions typically offer greater specificity for individual CIPK proteins

  • The NAF/FISL motif that mediates CBL binding is conserved across CIPKs

• Cross-reactivity patterns:

  • Antibodies against CIPK9 may cross-react with CIPK19 and vice versa

  • The degree of cross-reactivity often correlates with sequence similarity

  • Validation against multiple recombinant CIPK proteins is essential

• Application-specific performance:

  • Some antibodies perform well in Western blot but poorly in immunoprecipitation

  • Fixation sensitivity varies for immunohistochemistry applications

  • Native protein recognition capability differs for interaction studies

• Evolutionary considerations:

  • CIPK proteins have evolved distinct functions while maintaining structural similarity

  • Species-specific variations may affect antibody recognition across plant species

When selecting or developing CIPK19 antibodies, researchers should consider these factors to ensure specificity and reliability for their specific applications.

What insights can be gained from comparing CIPK19 structure and function across different plant species?

Comparative analysis of CIPK19 across species provides valuable insights:

• Structural conservation:

  • The kinase domain typically shows higher conservation

  • Regulatory regions may diverge more rapidly between species

  • CBL-binding motifs are generally conserved but may show species-specific variations

• Functional evolution:

  • Some species may utilize CIPK19 for specific stress responses

  • Gene duplication events may lead to functional specialization

  • Expression patterns and tissue specificity can vary across species

• Antibody cross-species utility:

  • Antibodies targeting conserved epitopes will work across related species

  • Species-specific regions may require custom antibodies

  • Validation in each species of interest is recommended

• Evolutionary adaptations:

  • Desert plants may show adaptations in CIPK signaling for drought tolerance

  • Crop species may have domestication-related changes in CIPK function

  • Wild relatives often provide insights into evolutionary selection pressures

Understanding these variations can guide experimental design and interpretation when studying CIPK19 in different model systems or crop species.