CIPK23 Antibody

What is CIPK23 and what are its primary functions in plants?

CIPK23 is a serine/threonine protein kinase that interacts with calcineurin B-like (CBL) calcium sensors to regulate ion transport processes in plants. CIPK23 functions as a central regulator in several key physiological processes:

• Promotion of blue light-dependent stomatal opening through activation of inward-rectifying K+ channels (K+in)

• Positive regulation of potassium uptake through activation of the potassium channel AKT1

• Regulation of nitrate transport by modulating NPF6;3 (the most abundant nitrate transporter)

• Inhibition of high-affinity ammonium transporters (AMT1s) through phosphorylation

These multiple roles position CIPK23 as a master regulator of ion homeostasis and environmental responses in plants.

Which plant species have characterized CIPK23 proteins suitable for antibody development?

CIPK23 has been most extensively characterized in Arabidopsis thaliana (AtCIPK23), where it mediates multiple physiological processes related to ion transport . Research has also identified and characterized CIPK23 in rice (Oryza sativa, OsCIPK23), particularly in the context of viral protein interactions . When developing or selecting CIPK23 antibodies, researchers should consider species-specific sequence variations that may affect epitope recognition and cross-reactivity.

How do CIPK23 protein expression patterns vary across plant tissues?

CIPK23 exhibits differential expression across plant tissues with particularly high levels detected in guard cell protoplasts (GCPs) compared to other tissues. RT-PCR analysis has confirmed CIPK23 transcript expression in:

• Guard cell protoplasts (highest expression)

• Mesophyll cell protoplasts

• Rosette leaves

• Petioles

• Inflorescence stems

• Roots

This tissue-specific expression pattern aligns with CIPK23's functional role in stomatal regulation and ion transport processes throughout the plant.

What experimental approaches can verify CIPK23 protein-protein interactions in planta?

Multiple complementary techniques have successfully demonstrated CIPK23 interactions with partner proteins:

• Bimolecular Fluorescence Complementation (BiFC): This technique has confirmed interactions between CIPK23 and phototropin proteins (phot1/phot2) in tobacco (Nicotiana benthamiana) leaves, visualizing interaction in living plant cells .

• In vitro pull-down assays: FLAG-tagged CIPK23 co-purifies with both phot1 and phot2 from microsomal membrane fractions, confirming their physical interaction .

• Yeast two-hybrid assays: This approach has demonstrated interactions between CIPK23 and ammonium transporters (AMT1;1 and AMT1;2) using multiple reporter systems (Ade2, His3, and β-galactosidase) .

• AlphaScreen methodology: This technique identified CIPK23 as a phototropin-interacting protein with binding affinity comparable to known interactors .

When designing interaction studies with CIPK23 antibodies, researchers should consider these validated approaches to ensure robust results.

How does CIPK23 phosphorylation status change in response to environmental stimuli?

CIPK23 regulation is dynamically responsive to environmental conditions, particularly nutrient availability:

When designing experiments to study CIPK23 phosphorylation, researchers should include appropriate time points (30 min, 1h, 2h) following environmental stimuli and use phospho-specific antibodies if available.

What are the implications of CIPK23 localization patterns for experimental design?

CIPK23 exhibits complex subcellular localization patterns that impact experimental approaches:

• Cytoplasmic and nuclear distribution: CIPK23 shows both cytoplasmic and nuclear localization in plant cells .

• Dynamic recruitment: Viral proteins (e.g., RGSV P1) can recruit CIPK23 to specific nuclear structures such as Cajal bodies, altering its normal localization pattern .

• Membrane association: CIPK23 interacts with membrane-associated proteins including phototropins and ion channels, suggesting transient membrane localization during signaling events .

When designing immunolocalization experiments, researchers should:

• Use cellular fractionation techniques to enrich different subcellular compartments

• Consider fixation methods that preserve both soluble and membrane-associated CIPK23 pools

• Include co-localization studies with validated markers for various cellular compartments

What are the optimal protein extraction protocols for CIPK23 immunoprecipitation?

For successful immunoprecipitation of CIPK23 from plant tissues, consider the following protocol recommendations:

• Extraction buffer composition:

  • Tris-HCl (50 mM, pH 7.5)

  • NaCl (150 mM)

  • EDTA (1 mM)

  • Triton X-100 (0.5-1%)

  • Protease inhibitor cocktail

  • Phosphatase inhibitor cocktail (critical for preserving phosphorylation status)

• Tissue preparation:

  • Flash-freeze tissue in liquid nitrogen

  • Grind thoroughly to fine powder while maintaining freezing temperature

  • Use guard cell-enriched preparations for higher CIPK23 yield

• Membrane protein considerations:

  • Include membrane solubilization steps for complete extraction of CIPK23 pools interacting with membrane proteins

  • Consider microsomal membrane fractionation as described for phototropin-CIPK23 interaction studies

How can researchers address challenges in detecting native CIPK23 protein levels?

Native CIPK23 detection presents several challenges that researchers should address through:

• Antibody validation strategies:

  • Verify specificity using cipk23 mutant lines as negative controls

  • Test antibody cross-reactivity with recombinant CIPK23 protein

  • Evaluate specificity across closely related CIPK family members

• Signal enhancement approaches:

  • Employ tissue-specific enrichment (e.g., guard cell protoplast isolation) where CIPK23 expression is highest

  • Use sensitive detection methods such as chemiluminescence with signal enhancers

  • Consider protein concentration steps before immunoblotting

• Quantification methods:

  • Use internal loading controls optimized for plant tissues

  • Implement fluorescent secondary antibodies for more accurate quantification

  • Develop appropriate normalization strategies for cross-tissue comparisons

What experimental designs effectively demonstrate CIPK23's dual regulation of K+ and NH4+ transport?

To investigate CIPK23's role in regulating both potassium and ammonium transport, researchers should consider the following experimental designs:

For comprehensive analysis, combine these approaches with molecular assays:

• Co-immunoprecipitation of CIPK23 with ion transporters using CIPK23 antibodies

• Phosphorylation assays to detect CIPK23-mediated modification of transport proteins

• In vivo transport activity measurements in heterologous expression systems

How can CIPK23 antibodies help elucidate blue light-dependent stomatal opening mechanisms?

CIPK23 antibodies can be employed to investigate several aspects of the phototropin signaling pathway in stomatal guard cells:

• Signaling complex formation:

  • Immunoprecipitate CIPK23 from blue light-treated guard cells to identify novel interaction partners

  • Analyze co-purifying proteins by mass spectrometry to map the signaling network

  • Track temporal changes in complex composition following blue light exposure

• Phototropin-CIPK23-K+in channel pathway:

  • Determine if CIPK23 directly phosphorylates K+in channels using in vitro kinase assays

  • Investigate the relationship between CIPK23 and other phototropin substrates like BLUS1

  • Analyze whether CIPK23 forms a complex with both phototropins and K+in channels simultaneously

• Physiological response correlation:

  • Quantify CIPK23 protein levels and phosphorylation status in parallel with stomatal aperture measurements

  • Compare wild-type and mutant responses to varying blue light intensities and duration

  • Determine how CIPK23 coordinates with H+-ATPase signaling pathways

What approaches can identify novel CIPK23 substrates in plant ion homeostasis?

To discover novel CIPK23 substrates involved in ion homeostasis regulation, researchers should consider these complementary approaches:

• Phosphoproteomic screening:

  • Compare phosphoproteomes of wild-type and cipk23 mutant plants under various ionic conditions

  • Analyze differentially phosphorylated proteins after induced CIPK23 expression

  • Focus on membrane proteins involved in ion transport

• Candidate-based approaches:

  • Test direct phosphorylation of ion transporters by CIPK23 in vitro

  • Examine transporters known to be regulated under conditions where CIPK23 is active

  • Investigate family members of confirmed CIPK23 substrates (e.g., other AMT proteins beyond AMT1;1 and AMT1;2)

• Interaction mapping:

  • Use CIPK23 antibodies for large-scale immunoprecipitation followed by mass spectrometry

  • Perform yeast two-hybrid screens with CIPK23 as bait against ion transporter libraries

  • Validate interactions in planta through BiFC and co-immunoprecipitation

How can CIPK23 antibodies aid in investigating pathogen-induced disruption of ion homeostasis?

Recent research revealing viral protein interactions with CIPK23 opens new avenues for studying pathogen interference with plant ion homeostasis:

• Pathogen-induced relocalization:

  • Track CIPK23 subcellular localization changes during pathogen infection using immunofluorescence

  • Investigate recruitment of CIPK23 to specific cellular compartments like Cajal bodies during viral infection

  • Compare the impact of different pathogens on CIPK23 distribution patterns

• Functional consequences analysis:

  • Measure ion transport activity in infected versus healthy tissues

  • Correlate CIPK23 sequestration with changes in K+ uptake efficiency

  • Examine stomatal responses in pathogen-infected plants

• Intervention strategies:

  • Identify the minimal interaction domains between viral proteins and CIPK23

  • Test peptide inhibitors that prevent pathogen-induced CIPK23 relocalization

  • Develop plant lines with modified CIPK23 that maintains function but resists pathogen manipulation