CIPK21 Antibody

What is CIPK21 and what is its role in plant stress responses?

CIPK21 is a CBL-interacting protein kinase ubiquitously expressed in plant tissues that plays a crucial role in mediating responses to salt and osmotic stress conditions in Arabidopsis thaliana. Research has demonstrated that CIPK21 is up-regulated under multiple abiotic stress conditions, including salt stress, mannitol treatment, abscisic acid exposure, cold conditions, and drought . Loss-of-function mutants (cipk21) exhibit hypersensitivity to high salt and osmotic stress conditions, indicating that CIPK21 functions as a positive regulator of salt and drought responses . The protein appears to regulate ion and water homeostasis across vacuolar membranes, particularly under salt stress conditions .

How does CIPK21 interact with other proteins in calcium signaling pathways?

CIPK21 physically interacts with calcium sensors CBL2 and CBL3 as demonstrated through yeast two-hybrid assays, in vivo localization studies, and bimolecular fluorescence complementation (BiFC) analysis . These calcium sensors target CIPK21 to the tonoplast (vacuolar membrane) . Stronger interactions were detected with CBL2 and CBL3, while weaker interactions were observed with CBL1 and CBL9 . This association with calcium sensors suggests that CIPK21 functions within the calcium-mediated signaling network that helps plants respond to environmental stresses .

What expression pattern does CIPK21 exhibit in Arabidopsis tissues?

CIPK21 shows a ubiquitous expression pattern across various plant tissues. Semiquantitative reverse transcription PCR analysis revealed expression in roots, stems, siliques, and flowers of 21-day-old Arabidopsis plants, as well as in 3-day-old seedlings during early development . GUS reporter assays with transgenic Arabidopsis plants harboring a CIPK21 promoter::β-glucuronidase construct showed strong expression in 1-day-old seedlings with a uniformly distributed pattern . In 10-day-old seedlings, expression was detected in open cotyledons and was less pronounced in roots while being undetectable in the hypocotyl . High expression was also observed in vascular tissues and root tips .

What are the recommended applications for CIPK21 antibodies in plant stress research?

CIPK21 antibodies serve multiple research applications in studying plant stress responses. For protein localization studies, they can be used in immunofluorescence microscopy to confirm the subcellular distribution of CIPK21, particularly its translocation to the tonoplast under salt stress conditions when co-expressed with CBL2 or CBL3 . In immunoprecipitation experiments, these antibodies can help isolate CIPK21-interacting protein complexes to identify novel partners beyond the known CBL interactions . For western blotting, CIPK21 antibodies enable quantification of protein expression levels across different tissues or in response to various stresses, complementing the transcript-level data already available . Additionally, chromatin immunoprecipitation (ChIP) assays using CIPK21 antibodies can help identify potential downstream targets if CIPK21 has nuclear functions.

How can CIPK21 antibodies be validated for specificity in Arabidopsis research?

Multiple validation approaches should be employed to ensure CIPK21 antibody specificity. The most definitive control is testing the antibody against cipk21 knockout/loss-of-function mutant tissues, which should show significantly reduced or absent signal compared to wild-type plants . Peptide competition assays, where the antibody is pre-incubated with the immunizing peptide before application to samples, can confirm binding specificity. For western blot validation, the antibody should detect a protein of the expected molecular weight (~58 kDa for CIPK21), with signal reduction in stress conditions known to affect protein levels . Cross-reactivity testing against recombinant proteins of closely related CIPKs (especially those with high sequence homology) is also recommended to ensure the antibody doesn't detect other CIPK family members.

What sample preparation techniques optimize CIPK21 detection with antibodies?

For optimal CIPK21 detection, sample preparation should preserve protein structure and phosphorylation state. Extraction buffers containing phosphatase inhibitors are essential since CIPK21 is a kinase subject to regulatory phosphorylation events . When isolating membrane-associated CIPK21 (especially after salt stress when it localizes to the tonoplast), detergent selection is critical—mild non-ionic detergents like 0.5-1% Triton X-100 or 1% NP-40 effectively solubilize membrane proteins while preserving antibody epitopes . For immunoprecipitation, native conditions with shorter extraction times at 4°C help maintain protein-protein interactions, particularly the CBL2/3-CIPK21 complexes . For fixed tissue samples, paraformaldehyde fixation (4%) followed by gentle permeabilization works well for maintaining the subcellular localization pattern observed in vivo .

How can CIPK21 antibodies be used to study its translocation under different stress conditions?

To study CIPK21 translocation under stress conditions, researchers can employ fractionation studies combined with immunoblotting using CIPK21 antibodies. This approach involves separating cytosolic, nuclear, and membrane fractions from plant cells under control and stress conditions, followed by western blotting with CIPK21-specific antibodies to quantify relative protein abundance in each compartment . Immunofluorescence microscopy offers another powerful method, where fixed plant cells are probed with CIPK21 antibodies and visualized with fluorescent secondary antibodies to track localization changes . The BiFC approach can be complemented with immunoprecipitation assays using CIPK21 antibodies to confirm the formation of CBL2/3-CIPK21 complexes under salt stress conditions and potentially identify additional interacting partners . Time-course experiments tracking CIPK21 localization at different time points after stress application can reveal the dynamics of this translocation process and correlate it with physiological responses .

What strategies address cross-reactivity issues when using CIPK21 antibodies in other plant species?

Cross-reactivity issues when using Arabidopsis CIPK21 antibodies in other plant species require strategic approaches. Researchers should first perform sequence alignment analysis of CIPK21 orthologs across species to identify conserved epitopes that might be recognized by existing antibodies . Western blot validation using recombinant CIPK21 proteins from both Arabidopsis and the target species can quantitatively assess cross-reactivity potential . For improved specificity, custom antibodies can be developed against conserved CIPK21 regions identified through bioinformatic analysis of multiple plant species . If significant sequence divergence exists, it may be necessary to develop species-specific antibodies targeting unique epitopes in the CIPK21 ortholog of interest . Alternatively, epitope-tagging approaches, where recombinant CIPK21 orthologs are expressed with common epitope tags in the target species, allow detection using well-characterized commercial tag antibodies rather than relying on cross-reactive CIPK21 antibodies .

How can phosphorylation-specific CIPK21 antibodies reveal activation mechanisms during stress responses?

Phosphorylation-specific CIPK21 antibodies offer powerful insights into activation mechanisms during stress responses. For development of these specialized antibodies, researchers should first identify the key regulatory phosphorylation sites in CIPK21 through mass spectrometry analysis of the protein isolated from plants under normal and stress conditions . Antibodies raised against phosphopeptides containing these sites can then specifically detect activated CIPK21 . Validation requires demonstrating that these antibodies recognize only phosphorylated CIPK21 and not the unphosphorylated form, using both recombinant proteins and plant samples treated with or without phosphatase . Time-course experiments tracking CIPK21 phosphorylation status after stress application can reveal the temporal dynamics of kinase activation in relation to physiological responses and calcium signaling events . Co-immunoprecipitation studies using these phospho-specific antibodies can identify proteins that interact specifically with the activated form of CIPK21, potentially revealing downstream targets .

Why might CIPK21 antibodies show variable results in different subcellular fractions?

Variability in CIPK21 antibody performance across subcellular fractions can stem from multiple factors. The dynamic localization of CIPK21 between cytosol and tonoplast depending on stress conditions and CBL interactions creates naturally varying concentrations that may appear as inconsistent detection . Extraction protocols significantly impact results—harsh detergents may denature the epitope in membrane fractions, while insufficient solubilization can reduce protein recovery from tonoplast-associated CIPK21 . Post-translational modifications, particularly phosphorylation states that change during stress responses, may alter epitope accessibility in different cellular compartments . The presence of interacting proteins like CBL2 and CBL3 can mask antibody binding sites when CIPK21 is in complex at the tonoplast . This issue appears more pronounced in tonoplast fractions under salt stress conditions when CIPK21 preferentially localizes there . Researchers should optimize extraction buffers with appropriate detergents (0.5-1% Triton X-100) and include phosphatase inhibitors to maintain epitope integrity across all fractions .

What control experiments are essential when using CIPK21 antibodies to study protein interactions?

When studying CIPK21 protein interactions, several control experiments are essential for reliable results. Negative controls should include cipk21 knockout/loss-of-function mutant tissues to confirm antibody specificity and establish background levels in co-immunoprecipitation experiments . Pre-clearing samples with non-immune IgG helps identify non-specific interactions that might otherwise be attributed to CIPK21 . Reciprocal co-immunoprecipitation, where both CIPK21 and its putative partner (e.g., CBL2 or CBL3) are immunoprecipitated in separate experiments, strengthens evidence for true interactions . Competition assays with recombinant CIPK21 can validate the specificity of detected interactions . Researchers should also include known interaction controls—since CIPK21 interaction with CBL2 and CBL3 is well-established, these serve as positive controls for co-immunoprecipitation procedures . Finally, comparing interactions under different conditions (with/without salt stress) is important since CIPK21 partnerships may be condition-dependent, as seen with its enhanced tonoplast localization during salt stress .

How can researchers overcome low endogenous CIPK21 detection issues in plant samples?

Overcoming low endogenous CIPK21 detection requires optimization at multiple levels. Researchers should consider tissue selection carefully, focusing on tissues with documented higher CIPK21 expression (e.g., root tips, vascular tissues, young seedlings) based on GUS reporter studies . Timing is also critical—harvesting tissues during peak expression periods or after stress induction when CIPK21 is upregulated improves detection . For extraction, using specialized buffers with protease inhibitors, reducing agents, and phosphatase inhibitors preserves CIPK21 protein integrity and abundance . Signal amplification techniques like using high-sensitivity ECL substrates for western blots or tyramide signal amplification for immunofluorescence can enhance detection of low-abundance proteins . Researchers might also consider protein concentration methods such as immunoprecipitation followed by western blotting to enrich for CIPK21 before detection . If these approaches prove insufficient, transgenic lines with epitope-tagged CIPK21 under native promoter control provide an alternative that maintains physiological expression patterns while enabling detection with highly-specific tag antibodies .

How does CIPK21 kinase activity correlate with its subcellular localization during stress responses?

CIPK21 kinase activity appears to be closely linked with its subcellular localization during stress responses. Research demonstrates that CIPK21 exhibits dynamic localization, shifting from a primarily cytosolic and nuclear distribution under normal conditions to increased tonoplast association during salt stress, particularly when co-expressed with CBL2 or CBL3 . This translocation likely represents an activation mechanism, whereby calcium signals detected by CBL2/3 facilitate CIPK21 recruitment to the vacuolar membrane where its substrates may reside . Bimolecular fluorescence complementation (BiFC) analysis revealed enhanced tonoplast association of CBL2/3-CIPK21 complexes specifically under salt stress conditions . This spatial regulation suggests that CIPK21 kinase activity is directed toward tonoplast-localized targets during osmotic stress, potentially phosphorylating ion channels or transporters to regulate ion and water homeostasis across vacuolar membranes . The functional significance of this localization-dependent activity is supported by the salt and osmotic stress hypersensitivity observed in cipk21 loss-of-function mutants .

What methodological approaches can identify downstream phosphorylation targets of CIPK21?

Identifying CIPK21 downstream phosphorylation targets requires multi-faceted approaches. Phosphoproteomic analyses comparing wild-type and cipk21 mutant plants under control and stress conditions can reveal differential phosphorylation patterns dependent on CIPK21 function . Researchers can employ in vitro kinase assays using purified recombinant CIPK21 with candidate substrates predicted through bioinformatic analyses or isolated tonoplast protein fractions, followed by mass spectrometry to identify phosphorylated residues . Yeast two-hybrid screens using CIPK21 as bait can identify interacting proteins that might represent substrates, with interactions subsequently validated through co-immunoprecipitation with CIPK21 antibodies . For targeted approaches, known tonoplast transporters and channels can be tested as CIPK21 substrates given the protein's stress-induced tonoplast localization . Genetic approaches comparing the phenotypes of cipk21 mutants with mutants of potential target proteins under stress conditions can provide functional evidence for regulatory relationships . Finally, proximity-dependent labeling techniques like BioID using CIPK21 fusions can identify proteins in close proximity at the tonoplast during stress responses .

How does the CIPK21 interactome change during different phases of the stress response?

The CIPK21 interactome likely undergoes dynamic changes throughout different phases of stress response. During early stress perception, calcium signals trigger initial formation of CBL2/3-CIPK21 complexes, as demonstrated by BiFC analyses showing enhanced tonoplast association under salt stress . Time-course co-immunoprecipitation experiments using CIPK21 antibodies at various time points after stress application would reveal the temporal dynamics of these interactions . In the adaptation phase, CIPK21 likely forms complexes with substrate proteins at the tonoplast, particularly transporters or channels involved in ionic and osmotic homeostasis across vacuolar membranes . Quantitative interaction proteomics comparing CIPK21 binding partners between early stress perception, adaptation, and recovery phases would provide a comprehensive view of these dynamic changes . The recovery phase may involve interactions with phosphatases like AP2C1, which has been shown to interact with related CIPKs . This interaction could potentially deactivate CIPK21 through dephosphorylation, though direct evidence for AP2C1-CIPK21 interaction is not established in the provided search results . Phosphorylation-dependent interaction studies would further reveal how CIPK21's activation state influences its interactome throughout the stress response cycle .

How do researchers reconcile conflicting data on CIPK21 subcellular localization across different studies?

Conflicting data on CIPK21 subcellular localization can be reconciled through careful examination of experimental conditions and methodologies. The search results indicate that when expressed alone, CIPK21 exhibits cytosolic and nuclear localization, but when co-expressed with CBL2 or CBL3, it shows additional tonoplast association that increases under salt stress . These seemingly contradictory observations actually represent context-dependent localization rather than true contradictions . Technical factors like expression systems (transient versus stable), fusion proteins (N-terminal versus C-terminal tags), expression levels (overexpression versus endogenous), and detection methods (direct fluorescence versus antibody-based) can all influence localization results . Studies using physiological expression levels with native promoters may better reflect true in vivo localization than overexpression systems . Additionally, the dynamic nature of CIPK21 localization during stress responses means that sampling time points and precise stress conditions significantly impact observations . To reconcile conflicting data, researchers should directly compare localization patterns under standardized conditions using multiple complementary approaches—fluorescent protein fusions, immunolocalization with CIPK21 antibodies, and biochemical fractionation—while carefully controlling for all variables .

What are the current limitations in studying CIPK21 post-translational modifications with available antibodies?

Current limitations in studying CIPK21 post-translational modifications (PTMs) with available antibodies present significant challenges. Standard CIPK21 antibodies typically recognize epitopes regardless of modification state, making them unsuitable for distinguishing between phosphorylated and non-phosphorylated forms of the protein . This limitation obscures critical regulatory information, as kinase activation often depends on specific phosphorylation events . The dynamic nature of PTMs during stress responses requires temporal resolution that general antibodies cannot provide . The potentially low abundance of modified CIPK21 forms presents detection challenges even with highly specific antibodies . Additionally, the search results don't indicate the availability of modification-specific CIPK21 antibodies, suggesting a significant gap in available research tools . Alternative approaches like mass spectrometry can identify modifications but lack the convenience of antibody-based detection for routine experiments . To overcome these limitations, researchers need to develop phospho-specific antibodies targeting key regulatory sites in CIPK21 and employ enrichment strategies to concentrate modified forms before detection .

What research questions remain unanswered regarding CIPK21's role in plant stress tolerance mechanisms?

Several critical research questions about CIPK21's role in plant stress tolerance remain unanswered. The precise molecular mechanisms by which CIPK21 regulates ion and water homeostasis across vacuolar membranes are still unclear . The specific targets phosphorylated by CIPK21 at the tonoplast during stress responses have not been definitively identified in the provided search results . The functional significance of CIPK21's weaker interactions with CBL1 and CBL9 (compared to CBL2/3) remains unexplored, potentially representing additional regulatory mechanisms or cellular contexts . The cross-talk between CIPK21-mediated signaling and other stress response pathways, including potential integration with hormone signaling networks like ABA, requires further investigation . The search results mention CIPK21 upregulation in response to cold stress, but its functional role in cold tolerance hasn't been characterized as thoroughly as its role in salt/osmotic stress . The evolutionary conservation of CIPK21 function across different plant species and its potential applications for improving crop stress tolerance remain important areas for future research . Additionally, potential regulatory mechanisms controlling CIPK21 activity beyond CBL interactions, such as phosphorylation by upstream kinases or dephosphorylation by phosphatases like AP2C1, warrant further study .