CIPK24 Antibody

What is CIPK24 and why is it important in plant research?

CIPK24 is a serine/threonine protein kinase that plays a critical role in plant salt stress response pathways. It contains an N-terminal kinase domain and a C-terminal regulatory region with an auto-inhibitory domain. The importance of CIPK24 stems from its central role in the Salt Overly Sensitive (SOS) pathway, where it functions as a key regulatory component mediating salt tolerance.

In Arabidopsis, CIPK24 (also known as SOS2) is activated by the calcium sensor CBL4 (SOS3) in response to salt stress. The activated CIPK24 then phosphorylates the plasma membrane Na⁺/H⁺ antiporter SOS1, enhancing Na⁺ efflux from root cells and contributing to salt tolerance . Beyond this, CIPK24 has been found to target tonoplast Na⁺/H⁺ antiporters and vacuolar H⁺-ATPase, increasing Na⁺ sequestration in vacuoles and thus reducing cytosolic Na⁺ toxicity . These functions make CIPK24 an important research target for understanding and potentially enhancing crop salt tolerance.

How do CIPK24 antibodies differ from other research antibodies?

CIPK24 antibodies are specifically designed to recognize and bind to CIPK24 protein epitopes, making them distinct from other research antibodies in several ways:

First, CIPK24 antibodies must be highly specific to distinguish CIPK24 from other closely related CIPK family members, which share significant sequence homology. This requires careful epitope selection during antibody development, typically targeting unique regions of CIPK24 that differ from other CIPKs.

Second, since CIPK24 exists in different activation states (inactive with the auto-inhibitory domain blocking the kinase domain, and active when this inhibition is released by CBL4 binding or phosphorylation at Thr168), researchers may need specific antibodies that can distinguish between these conformational states .

Third, CIPK24 antibodies need to function effectively in plant tissue samples, which can present different challenges compared to mammalian samples due to differences in protein abundance, extraction conditions, and the presence of plant-specific compounds that may interfere with antibody binding.

Finally, since CIPK24 interacts with multiple partners including CBL4, SOS1, and other proteins, antibodies must be designed not to interfere with these interaction sites if they are to be used in co-immunoprecipitation or other interaction studies .

What are the primary research applications for CIPK24 antibodies?

CIPK24 antibodies serve multiple essential functions in plant stress biology research:

• Protein Detection and Quantification: Western blotting using CIPK24 antibodies allows researchers to detect and quantify CIPK24 protein expression levels in different plant tissues, under varying stress conditions, or across different genotypes.

• Immunolocalization: Immunofluorescence microscopy with CIPK24 antibodies enables visualization of the subcellular localization of CIPK24. This is particularly important since CIPK24 may relocalize from the cytosol to the plasma membrane upon activation, as demonstrated in BiFC assays showing CIPK24-CBL4 interactions at the plasma membrane .

• Protein-Protein Interaction Studies: CIPK24 antibodies are crucial for co-immunoprecipitation experiments to identify and verify CIPK24 interaction partners. This has been instrumental in elucidating how CIPK24 interacts with calcium sensors like CBL4 in a Ca²⁺-dependent manner .

• Phosphorylation State Detection: Specialized phospho-specific antibodies can detect the phosphorylation state of CIPK24, particularly at the Thr168 residue in the activation loop, which is critical for monitoring CIPK24 activation status.

• Chromatin Immunoprecipitation (ChIP): Although CIPK24 itself is not a transcription factor, CIPK24 antibodies can be used in ChIP experiments when studying protein complexes that might influence gene expression during salt stress responses.

How should researchers design experiments to validate CIPK24 antibody specificity?

Validating CIPK24 antibody specificity is crucial for reliable experimental results. A comprehensive validation approach should include:

• Western Blot Analysis with Positive and Negative Controls:

  • Positive control: Recombinant CIPK24 protein or extracts from plants overexpressing CIPK24

  • Negative control: Extracts from cipk24 knockout/knockdown plants

  • Comparative analysis: Test the antibody against closely related CIPK family members to verify lack of cross-reactivity

• Epitope Competition Assay: Pre-incubate the antibody with excess purified epitope peptide before immunodetection. Disappearance of the CIPK24 signal confirms specificity to the target epitope.

• Immunoprecipitation Followed by Mass Spectrometry: Perform immunoprecipitation with the CIPK24 antibody and analyze the purified proteins by mass spectrometry to confirm CIPK24 identity and detect any cross-reacting proteins.

• Testing in Multiple Plant Species: If the antibody is designed to recognize CIPK24 across species (such as both Arabidopsis CIPK24 and tomato SlCIPK24), verify specificity in each species. The sequence similarity between these orthologs supports potential cross-reactivity, as both proteins can interact with their counterpart CBL4 proteins across species .

• Antibody Performance Under Different Experimental Conditions: Test the antibody under various extraction and detection conditions, as protein denaturation state can affect epitope accessibility.

What is the optimal protocol for using CIPK24 antibodies in co-immunoprecipitation experiments?

For successful co-immunoprecipitation (Co-IP) of CIPK24 and its interacting partners:

• Sample Preparation:

  • Extract proteins from plant tissues using a gentle, non-denaturing buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitor cocktail)

  • Consider adding phosphatase inhibitors to maintain phosphorylation status

  • When studying Ca²⁺-dependent interactions like CIPK24-CBL4, carefully control Ca²⁺ levels in the buffer as this interaction may be Ca²⁺-dependent, unlike some Arabidopsis CBL-CIPK interactions that form complexes without Ca²⁺

• Pre-clearing:

  • Pre-clear lysate with protein A/G beads to reduce non-specific binding

  • Reserve a small sample of the input for later analysis

• Immunoprecipitation:

  • Incubate pre-cleared lysate with CIPK24 antibody (typically 2-5 μg per 500 μg protein) overnight at 4°C

  • Add pre-washed protein A/G beads and incubate for 2-4 hours

  • Wash beads thoroughly (at least 4-5 times) with buffer containing reduced detergent

• Elution and Analysis:

  • Elute proteins by boiling in SDS sample buffer

  • Analyze by SDS-PAGE followed by western blotting with antibodies against potential interacting proteins (e.g., CBL4, SOS1)

  • For the detection of the NAF/FISL motif-dependent interactions between CIPK24 and CBL4, ensure that the C-terminal region of CIPK24 containing this motif is accessible

• Controls:

  • Negative control: Perform parallel Co-IP with non-specific IgG

  • Verification: Reverse Co-IP using antibodies against interacting partners

  • For studying interactions similar to the SlCBL4-SlCIPK24 complex, consider using deletion mutants like SlCIPK24N (lacking the NAF motif) as negative controls

What experimental considerations are important when using CIPK24 antibodies to study protein localization?

When using CIPK24 antibodies for immunolocalization studies:

• Fixation and Sample Preparation:

  • Test multiple fixation methods (e.g., paraformaldehyde, methanol) as these can affect epitope accessibility

  • For plant tissues, cell wall digestion or permeabilization requires optimization to maintain tissue integrity while allowing antibody access

  • Perform antigen retrieval if necessary, especially for formaldehyde-fixed samples

• Blocking and Antibody Incubation:

  • Use appropriate blocking agents (BSA, normal serum) to reduce background

  • Optimize primary antibody dilution (typically starting with 1:100 to 1:500)

  • Incubate at 4°C overnight for best results

  • Include washing steps with PBS-T (PBS with 0.1% Tween-20)

• Controls and Verification:

  • Negative control: Omit primary antibody or use pre-immune serum

  • Positive control: Use tissues known to express CIPK24 (e.g., root tissues under salt stress)

  • Complementary approaches: Verify localization results with GFP-tagged CIPK24 expression or BiFC assays, similar to those used for SlCBL4-SlCIPK24 interaction studies

• Co-localization Studies:

  • Use markers for subcellular compartments (plasma membrane, cytosol, etc.)

  • For co-localization with interaction partners like CBL4, use double immunolabeling

  • Compare localization under normal and salt stress conditions, as CIPK24 may relocalize during stress

• Microscopy Considerations:

  • Use confocal microscopy for precise subcellular localization

  • For quantitative analysis, maintain consistent microscopy settings across samples

  • Consider super-resolution techniques for detailed subcellular localization studies

How can researchers modify standard protocols to detect phosphorylated forms of CIPK24?

Detecting phosphorylated CIPK24 requires specialized approaches:

• Phospho-specific Antibodies:

  • Use antibodies specifically raised against phosphorylated Thr168 in the activation loop of CIPK24

  • Validate phospho-antibody specificity using recombinant CIPK24 treated with phosphatases as negative controls

  • Consider creating a superactive SlCIPK24 mutant (SlCIPK24M) with a Thr168 to Asp substitution as a positive control, similar to the approach used in Arabidopsis

• Phosphatase Treatment Controls:

  • Split samples and treat half with lambda phosphatase to confirm signal is phosphorylation-dependent

  • Include phosphatase inhibitors in protein extraction buffers to preserve phosphorylation status

• Phos-tag™ SDS-PAGE:

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

  • Run proteins longer than in standard SDS-PAGE to achieve optimal separation

  • Follow with western blotting using regular CIPK24 antibodies

• 2D Gel Electrophoresis:

  • Separate proteins first by isoelectric point (affected by phosphorylation) then by molecular weight

  • Detect with standard CIPK24 antibodies

  • Compare patterns with and without phosphatase treatment

• Mass Spectrometry:

  • Immunoprecipitate CIPK24 using standard antibodies

  • Analyze by MS/MS to identify phosphorylation sites

  • For quantitative analysis, consider SILAC or TMT labeling approaches

What are common problems when using CIPK24 antibodies and how can they be resolved?

Researchers frequently encounter these challenges when working with CIPK24 antibodies:

• Weak or No Signal in Western Blots:

  • Problem: Low CIPK24 abundance in samples or poor antibody sensitivity

  • Solutions:

    • Enrich samples through immunoprecipitation before western blotting

    • Use enhanced chemiluminescence substrates or more sensitive detection methods

    • Increase antibody concentration or incubation time

    • Try different extraction buffers to improve CIPK24 solubilization

• Multiple Bands or Non-specific Binding:

  • Problem: Antibody cross-reactivity with other CIPK family members

  • Solutions:

    • Increase washing stringency (higher salt concentration or detergent)

    • Optimize blocking conditions

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

    • Use cipk24 mutant extracts to identify non-specific bands

• Inconsistent Results Between Experiments:

  • Problem: Variability in CIPK24 expression or extraction efficiency

  • Solutions:

    • Standardize plant growth conditions, especially regarding salt treatment

    • Use consistent protein extraction protocols

    • Include loading controls and normalization standards

    • Prepare larger batches of protein samples for multiple experiments

• Poor Immunoprecipitation Efficiency:

  • Problem: Inefficient CIPK24 capture during Co-IP

  • Solutions:

    • Optimize antibody-to-sample ratio

    • Try different antibody attachment methods (direct coupling to beads vs. protein A/G)

    • Adjust buffer conditions, particularly considering the Ca²⁺-dependence of some CIPK24 interactions

    • Consider crosslinking to capture transient interactions

• Background in Immunolocalization:

  • Problem: High background obscuring specific CIPK24 signal

  • Solutions:

    • Optimize blocking (try different agents: BSA, normal serum, commercial blockers)

    • Increase washing duration and number of washes

    • Titrate primary and secondary antibody concentrations

    • Try different fixation and permeabilization methods

How can researchers apply CIPK24 antibodies in studying CIPK24 activation mechanisms?

To investigate CIPK24 activation mechanisms:

• Monitoring Phosphorylation Status:

  • Use phospho-specific antibodies against Thr168 to track activation

  • Compare phosphorylation levels under different stress conditions and timepoints

  • Correlate phosphorylation with kinase activity assays using immunoprecipitated CIPK24

• Studying Conformational Changes:

  • Develop conformation-specific antibodies that recognize either the active or inactive form

  • Use limited proteolysis followed by immunoblotting to detect conformational states

  • Combine with structural approaches like hydrogen-deuterium exchange mass spectrometry

• Tracking Protein-Protein Interactions:

  • Use CIPK24 antibodies in Co-IP experiments under different Ca²⁺ conditions to assess Ca²⁺-dependency of interactions

  • Compare wild-type CIPK24 with mutant versions (e.g., NAF motif mutants) to elucidate interaction mechanisms

  • Combine with proximity labeling techniques (BioID, APEX) to identify transient interaction partners

• In vitro Reconstitution Experiments:

  • Immunoprecipitate CIPK24 from plants under different conditions

  • Assess its ability to phosphorylate substrates like SOS1 in vitro

  • Compare with recombinant CIPK24 activated by different means (CBL4 addition, Thr168 phosphomimetic mutation)

• Time-course Analysis During Stress Responses:

  • Track CIPK24 modifications, interactions, and localization over time following salt stress

  • Correlate with physiological responses like Na⁺ flux measurements

  • Compare wild-type plants with those expressing constitutively active CIPK24 forms, such as SlCIPK24M with enhanced salt tolerance

How should researchers interpret variability in CIPK24 detection levels across different plant tissues?

Variability in CIPK24 detection requires careful interpretation:

• Tissue-Specific Expression Patterns:

  • CIPK24 expression varies naturally across tissues, with typically higher expression in roots compared to shoots, correlating with its role in Na⁺ extrusion from root cells

  • Normalize detection to appropriate housekeeping proteins specific to each tissue type

  • Compare relative changes within the same tissue type rather than absolute levels across different tissues

• Technical vs. Biological Variability:

  • Technical variability: Run multiple technical replicates and standardize protein extraction protocols

  • Biological variability: Increase biological replicates (n≥3) and control growth conditions tightly

  • Use statistical methods appropriate for the data distribution (parametric or non-parametric)

• Extraction Efficiency Considerations:

  • Different tissues may require modified extraction protocols to achieve comparable efficiency

  • Consider using recombinant CIPK24 spiked into samples as an extraction control

  • Evaluate total protein recovery alongside specific CIPK24 detection

• Antibody Performance Across Tissues:

  • Antibody access may vary between dense (mature leaves) and less dense (young leaves, roots) tissues

  • Optimize extraction and immunodetection protocols for each tissue type

  • Use complementary methods (e.g., RT-qPCR) to correlate protein data with transcript levels

• Physiological Interpretation:

  • Consider how developmental stage and environmental conditions affect CIPK24 levels

  • Interpret differences in the context of tissue-specific salt stress responses

  • Note that protein levels may not directly correlate with activity levels due to post-translational regulation

What approaches can help distinguish between direct and indirect effects when studying CIPK24 function using antibodies?

Distinguishing direct from indirect effects requires multiple complementary approaches:

• In Vitro Kinase Assays:

  • Immunoprecipitate CIPK24 using specific antibodies

  • Test direct phosphorylation of purified substrates in vitro

  • Include controls with kinase-dead CIPK24 mutations

  • Identify phosphorylation sites by mass spectrometry

• Temporal Analysis:

  • Perform time-course studies to establish sequence of events

  • Early events (minutes to hours) are more likely to be direct effects

  • Use inducible expression systems to trigger CIPK24 activity and monitor immediate responses

• Genetic Approaches in Combination with Antibody Studies:

  • Compare cipk24 knockout/knockdown plants with wild-type and complemented lines

  • Use phosphorylation site mutants of potential substrates to confirm direct relationships

  • Create plants expressing constitutively active CIPK24 forms like the superactive SlCIPK24M

• Inhibitor Studies:

  • Use specific kinase inhibitors to block CIPK24 activity

  • Monitor which effects are rapidly reversed upon inhibition

  • Control for inhibitor specificity with resistant CIPK24 mutants

• Proximity-dependent Labeling:

  • Fuse CIPK24 to BioID or APEX2 enzymes

  • Identify proteins in close proximity during salt stress

  • Validate direct interactions with co-immunoprecipitation using CIPK24 antibodies

How can researchers reconcile contradictory findings between CIPK24 antibody-based studies and other experimental approaches?

When facing contradictory results:

• Methodological Differences Analysis:

  • Compare antibody specificity, sensitivity, and epitope locations

  • Assess differences in experimental conditions (plant age, stress treatment, timing)

  • Consider differences in genetic backgrounds or plant species used

  • Evaluate how protein extraction methods might affect detected CIPK24 pools

• Complementary Method Verification:

  • Validate antibody-based findings with genetic approaches (mutants, RNAi, overexpression)

  • Confirm localization studies with fluorescently-tagged proteins

  • Support interaction studies with yeast two-hybrid or BiFC assays

  • Verify expression data with transcript analysis

• Context-Dependent Function Consideration:

  • CIPK24 may exhibit different functions depending on:

    • Cell/tissue type (root cells vs. shoot cells)

    • Developmental stage

    • Stress conditions and duration

    • Presence of different interaction partners

  • Document all contextual variables carefully

• Technical Limitations Assessment:

  • Antibodies may not distinguish between different modified forms

  • Some interactions may be transient or occur in specific microenvironments

  • Consider detection thresholds of different methods

  • Evaluate whether tags (GFP, FLAG) might affect protein function

• Integrative Data Analysis:

  • Develop models that incorporate findings from multiple approaches

  • Weight evidence based on methodological strength

  • Use systems biology approaches to place CIPK24 in broader signaling networks

  • Consider that seemingly contradictory results might reveal new biological insights

How might new antibody technologies improve CIPK24 research?

Emerging antibody technologies offer new possibilities for CIPK24 research:

• Single-domain Antibodies (Nanobodies):

  • Smaller size allows better tissue penetration and epitope access

  • Can recognize conformational epitopes more effectively

  • May be expressed in vivo as intrabodies to track or modulate CIPK24 activity

  • Could potentially distinguish between active and inactive CIPK24 conformations

• Recombinant Antibody Fragments:

  • Fab or scFv fragments with reduced background in plant tissues

  • Site-specific labeling for super-resolution microscopy

  • Multiplexed detection with different fluorophores

  • Improved batch-to-batch consistency compared to polyclonal antibodies

• Bispecific Antibodies:

  • Target CIPK24 and interaction partners simultaneously

  • Study protein complexes in their native environment

  • Enable novel proximity-based detection methods

  • May be tailored for specific applications in plant research

• Phospho-state Specific Confirmation Sensors:

  • Antibodies designed to recognize specific active conformations

  • Monitor activation state in real-time in living cells

  • Could distinguish between different activation mechanisms (Ca²⁺-dependent vs. phosphorylation-dependent)

• Antibody-enabled Proteomics:

  • Antibody-based enrichment for targeted proteomics

  • Identification of post-translational modifications and interaction partners

  • Spatial proteomics to analyze CIPK24 complexes in different subcellular locations

  • Quantitative analysis of CIPK24 dynamics during stress responses

What are promising strategies for studying CIPK24 protein dynamics using antibodies?

Advanced approaches for investigating CIPK24 dynamics include:

• Single-molecule Tracking with Antibody Fragments:

  • Label CIPK24 with fluorescent antibody fragments

  • Track movement between cytoplasm and membrane in response to Ca²⁺ signals

  • Analyze diffusion rates and residence times at the plasma membrane

  • Correlate with salt stress responses

• Fluorescence Recovery After Photobleaching (FRAP):

  • Use fluorescently tagged antibodies to label CIPK24

  • Photobleach specific cellular regions and monitor recovery

  • Determine mobile vs. immobile fractions of CIPK24

  • Compare dynamics under different stress conditions

• Proximity Ligation Assay (PLA):

  • Detect CIPK24 interactions with partners like CBL4

  • Visualize interaction sites within cells

  • Quantify interaction events during stress response

  • Compare with BiFC results for SlCBL4-SlCIPK24 interaction at the plasma membrane

• Super-resolution Microscopy with Antibodies:

  • Apply STORM or PALM techniques with antibody-based labeling

  • Achieve nanometer-scale resolution of CIPK24 localization

  • Study co-localization with interacting proteins at unprecedented detail

  • Track relocalization during salt stress response

• Optogenetic Control Combined with Antibody Detection:

  • Engineer light-controlled CIPK24 activation

  • Monitor consequences using antibody-based detection

  • Create precise temporal activation maps

  • Correlate with physiological responses to salt stress

How can researchers use CIPK24 antibodies to study conservation and divergence across plant species?

Antibody-based comparative studies provide insights into CIPK24 evolution:

• Cross-reactivity Assessment:

  • Test antibodies raised against one species (e.g., Arabidopsis CIPK24) on proteins from other species

  • Map conserved epitopes using sequence alignment and epitope mapping

  • Develop species-specific antibodies for divergent regions

  • Compare molecular weights, modification patterns, and expression levels across species

• Functional Conservation Studies:

  • Use antibodies to compare subcellular localization patterns across species

  • Assess interaction with CBL4 orthologs (e.g., SlCBL4 in tomato) via co-immunoprecipitation

  • Compare phosphorylation patterns and sites between species

  • Correlate with salt tolerance phenotypes across species

• Expression Pattern Comparison:

  • Analyze tissue-specific expression patterns in different plant species

  • Compare induction kinetics during salt stress

  • Assess developmental regulation across species

  • Correlate with ecological adaptation to saline environments

• Heterologous Expression Studies:

  • Express CIPK24 from one species in another

  • Use antibodies to confirm expression and proper localization

  • Assess functionality through complementation of cipk24 mutants

  • Study potential dominant-negative effects

• Antibody Epitope Mapping:

  • Identify conserved vs. variable regions between orthologs

  • Generate species-specific antibodies targeting divergent regions

  • Map functional domains through correlation with activity assays

  • Compare with bioinformatic predictions of conserved motifs

This comprehensive FAQ collection covers fundamental aspects of CIPK24 antibody applications in research while providing methodologically detailed answers that address both basic and advanced research questions. The focus remains on academic research scenarios, avoiding commercial aspects, and emphasizing experimental design, troubleshooting, and data interpretation relevant to plant stress biology researchers.