CIPK25 Antibody

What is CIPK25 and why is it significant in plant research?

CIPK25 is a member of the calcineurin B-like interacting protein kinase family that functions as a calcium-regulated protein kinase. It plays a crucial role in coordinating auxin and cytokinin signaling in root meristem development . Additionally, CIPK25 is involved in regulating potassium homeostasis under low-oxygen conditions through direct interaction with AKT1, the main inward rectifying potassium channel in plants . Its dual role in developmental processes and stress responses makes it an important target for understanding signaling networks in plants.

How do CIPK25 antibodies differ from other CIPK family antibodies?

CIPK25 antibodies are specifically generated against unique epitopes of the CIPK25 protein that distinguish it from other CIPK family members. While all CIPKs contain a conserved kinase domain, CIPK25 antibodies typically target either the variable C-terminal regulatory domain or unique peptide sequences to ensure specificity. When selecting a CIPK25 antibody, researchers should verify cross-reactivity testing against other CIPK proteins, especially CIPK23, which has functional overlap in potassium regulation pathways . Western blot validation using both wild-type and cipk25 mutant lines is essential to confirm specificity before experimental use.

What experimental techniques commonly employ CIPK25 antibodies?

CIPK25 antibodies are versatile tools employed in multiple techniques:

• Western blotting: For detecting CIPK25 protein expression in various tissues and under different treatment conditions

• Immunoprecipitation: To isolate CIPK25 and its interacting proteins

• Immunolocalization: For visualizing subcellular localization of CIPK25

• Chromatin immunoprecipitation (ChIP): When studying transcription factors that might regulate CIPK25 expression

The selection of appropriate antibody depends on the specific technique. For example, immunolocalization studies may require antibodies with minimal background binding, while co-immunoprecipitation experiments benefit from antibodies that don't interfere with protein-protein interaction domains .

How should CIPK25 antibodies be validated before use in critical experiments?

Thorough validation of CIPK25 antibodies is crucial to ensure experimental reliability. A comprehensive validation protocol should include:

• Genetic validation: Testing antibody reactivity against protein extracts from wild-type plants versus cipk25 mutant lines (such as SALK_079011 or SALK_029271) and CIPK25-overexpression lines

• Peptide competition assay: Pre-incubating the antibody with the immunizing peptide to confirm binding specificity

• Cross-reactivity assessment: Testing against recombinant proteins of closely related CIPK family members

• Subcellular fractionation validation: Confirming detection in appropriate cellular compartments using fraction-specific markers (similar to those used in CIPK25 studies: anti-histone H3 for nuclear fraction, anti-plasma membrane H⁺ ATPase for membrane fraction, and anti-cytosolic fructose-1,6-bisphosphatase for cytosolic fraction)

This multi-step validation process ensures that experimental results truly reflect CIPK25 biology rather than artifacts or cross-reactivity.

What are the optimal conditions for using CIPK25 antibodies in Western blotting?

For optimal Western blotting results with CIPK25 antibodies, consider the following protocol adaptations:

• Sample preparation: Extract proteins from tissues with documented CIPK25 expression, particularly flowers and roots , using buffers containing phosphatase inhibitors to preserve phosphorylation status

• Protein separation: Use 10-12% SDS-PAGE gels to properly resolve the ~54 kDa CIPK25 protein

• Transfer conditions: Semi-dry transfer at 15V for 30 minutes or wet transfer at 30V overnight at 4°C

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

• Antibody incubation: Primary antibody dilution typically at 1:1000-1:2000, overnight at 4°C

• Signal detection: Enhanced chemiluminescence with exposure times optimized based on expression levels

When analyzing CIPK25 in different experimental conditions, include appropriate controls such as constitutively expressed proteins (actin, tubulin) to normalize loading and expression.

How can CIPK25 antibodies be utilized to study protein-protein interactions in calcium signaling networks?

CIPK25 antibodies are valuable tools for investigating protein-protein interactions within calcium signaling networks. Co-immunoprecipitation experiments can be designed to:

• Confirm known interactions: CIPK25 has been shown to interact with CBL4 and CBL5 in yeast two-hybrid assays . Antibodies can validate these interactions in plant tissues.

• Identify interaction conditions: Determine whether interactions are calcium-dependent by manipulating Ca²⁺ concentrations in binding buffers

• Map interaction domains: When combined with truncated protein constructs (such as CIPK25ΔC lacking the C-terminal domain) , antibodies can help map specific interaction regions

• Investigate dynamic interactions: Study how stimuli like auxin, cytokinin, or hypoxic conditions affect CIPK25 interactions with partners

A typical co-immunoprecipitation protocol would include:

• Tissue extraction in non-denaturing buffer

• Incubation with CIPK25 antibody coupled to protein A/G beads

• Washing to remove non-specific bindings

• Elution and analysis of bound proteins by Western blotting or mass spectrometry

How do CIPK25 antibodies enable the investigation of its dual function in hormone signaling and ion homeostasis?

CIPK25 uniquely functions in both auxin/cytokinin signaling pathways and potassium homeostasis under stress conditions. Antibodies enable researchers to investigate this dual functionality through:

• Differential expression analysis: Comparing CIPK25 protein levels in response to hormone treatments versus ion stress conditions

• Phosphorylation state assessment: Using phospho-specific antibodies to determine how different stimuli affect CIPK25 activation

• Subcellular localization studies: Tracking CIPK25 movement between cellular compartments using immunofluorescence microscopy

• Interactome shifts: Identifying stimulus-specific interaction partners through comparative immunoprecipitation

What approaches can resolve contradictory findings when using CIPK25 antibodies across different experimental systems?

When researchers encounter contradictory results with CIPK25 antibodies, several systematic approaches can help resolve discrepancies:

• Antibody epitope mapping: Different antibodies may recognize distinct regions of CIPK25 that could be masked in certain protein complexes or conformational states

• Post-translational modification analysis: Phosphorylation or other modifications may affect antibody recognition; use phosphatase treatments to test this hypothesis

• Tissue-specific expression patterns: CIPK25 shows differential expression across tissues, with notable absence in the cell proliferation domain of the root apical meristem

• Genetic background considerations: Ensure consistent genetic backgrounds when comparing results; the cipk25-2 (SALK_070911c) and cipk25-3 (SALK_059092) mutant lines have been well-characterized

• Stimulus conditions standardization: CIPK25 response varies with different stimuli; standardize treatment conditions when comparing across studies

How can CIPK25 antibodies contribute to understanding its role in oxidative stress responses?

Recent research suggests connections between CIPK family proteins and respiratory burst oxidase homologs (RBOHs) that generate reactive oxygen species. While CIPK6 has been shown to form a complex with CBL1/9 that negatively regulates RbohD , the potential role of CIPK25 in oxidative stress responses represents an emerging area of investigation. CIPK25 antibodies can contribute to this research through:

• Comparative co-immunoprecipitation: Determining whether CIPK25, like CIPK6, interacts with components of the ROS production machinery

• Stress-induced phosphorylation: Monitoring CIPK25 phosphorylation status during oxidative stress using phospho-specific antibodies

• Protein complex analysis: Identifying stress-specific interactors that might connect CIPK25 to ROS signaling networks

• In vitro kinase assays: Determining whether CIPK25 can phosphorylate RBOH proteins using purified components and immunoprecipitated CIPK25

Researchers should compare findings between cipk25 mutants and other cipk family mutants (particularly cipk6) to establish functional relationships in oxidative stress signaling pathways.

What methodological approaches can overcome challenges in detecting low-abundance CIPK25 protein in specific cell types?

Detecting low-abundance CIPK25 in specific cell types presents technical challenges that can be addressed through several methodological improvements:

• Signal amplification techniques:

  • Use tyramide signal amplification (TSA) with horseradish peroxidase-conjugated secondary antibodies

  • Apply multiplexed detection with quantum dot-conjugated antibodies

• Tissue-specific enrichment:

  • Employ laser capture microdissection to isolate specific cell types (particularly root endodermis where CIPK25 is induced under hypoxia)

  • Use fluorescence-activated cell sorting (FACS) with tissue-specific fluorescent markers

• Proximity ligation assay (PLA):

  • Detect protein-protein interactions with higher sensitivity than conventional co-immunoprecipitation

  • Particularly useful for studying CIPK25 interactions with CBLs or AKT1 in specific cell types

• Mass spectrometry enhancement:

  • Use sequential window acquisition of all theoretical mass spectra (SWATH-MS) for more sensitive detection

  • Apply targeted proteomics approaches like selected reaction monitoring (SRM)

These approaches can significantly improve detection sensitivity when conventional immunoblotting techniques fail to detect CIPK25 in specific cell types or under certain conditions.

What are common issues with CIPK25 antibodies and how can they be resolved?

Researchers frequently encounter specific challenges when working with CIPK25 antibodies. Here are common issues and their solutions:

• High background signal:

  • Increase blocking time/concentration (try 5% BSA instead of milk for phospho-detection)

  • Use more stringent washing conditions (increase salt concentration in wash buffer)

  • Pre-absorb antibody with plant extract from cipk25 mutant

• Weak or absent signal:

  • Enrich for membrane fractions where CIPK25 often localizes when interacting with ion channels

  • Use native lysis conditions to preserve epitope structure

  • Reduce denaturation temperature if the epitope is conformation-sensitive

• Multiple bands/non-specific binding:

  • Increase antibody dilution

  • Perform peptide competition assay to identify specific band

  • Use gradient gels to better resolve proteins of similar molecular weights

• Inconsistent results between experiments:

  • Standardize plant growth conditions (CIPK25 expression is affected by auxin, cytokinin, and oxygen levels)

  • Harvest tissues at consistent developmental stages

  • Use recombinant CIPK25 protein as a positive control in each experiment

How can researchers optimize immunoprecipitation protocols specifically for CIPK25 kinase activity assays?

Optimizing immunoprecipitation for subsequent CIPK25 kinase activity assays requires special considerations:

• Buffer composition optimization:

  • Use phosphate-free buffers to avoid interference with ATP during kinase assays

  • Include protease inhibitors but avoid phosphatase inhibitors if studying native phosphorylation state

  • Add 1-2 mM calcium to facilitate CBL-CIPK interactions during extraction

• Antibody selection and coupling:

  • Choose antibodies raised against regions distant from the kinase domain

  • Covalently couple antibodies to beads to prevent heavy chain contamination in eluates

  • Consider using tagged CIPK25 and anti-tag antibodies if native antibodies interfere with activity

• Washing conditions:

  • Use gentle washing to preserve protein-protein interactions

  • Include 0.1% non-ionic detergent to reduce non-specific binding

  • Maintain consistent salt concentration to preserve kinase activity

• Elution for activity preservation:

  • Elute with antibody-specific peptide rather than denaturing conditions

  • Perform kinase assays directly on bead-bound CIPK25 to minimize activity loss

  • Include CBL proteins (especially CBL1, which enhances CIPK6 kinase activity) in the reaction buffer

By implementing these optimizations, researchers can obtain more consistent and physiologically relevant results in CIPK25 kinase activity assays.