CIPK9 Antibody

What is CIPK9 and what are its main functions in plant systems?

CIPK9 (CBL-Interacting Protein Kinase 9) is a serine/threonine protein kinase that plays crucial roles in potassium (K+) homeostasis under low-K stress conditions in plants. It functions as a positive regulator of Arabidopsis root growth and seedling development under low-K+ conditions . CIPK9 interacts with the calcium sensor proteins CBL2 and CBL3, and this interaction is essential for its function in K+ deficiency response signaling pathways . Research has also revealed that CIPK9 is involved in ammonium sensing, as its mutation results in sensitivity to ammonium .

What methodological approaches are recommended for detecting CIPK9 in plant samples?

For detecting endogenous CIPK9 in plant tissues, immunoblotting using specific CIPK9 antibodies is the most direct approach. Begin with protein extraction using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5% glycerol, 1% Triton X-100, and protease inhibitor cocktail. After SDS-PAGE separation, transfer proteins to a PVDF membrane and probe with a specific anti-CIPK9 antibody (typically at 1:1000 dilution). For immunolocalization studies, fix tissue samples in 4% paraformaldehyde, embed in paraffin or resin, section, and perform immunolabeling using the CIPK9 antibody followed by a fluorophore-conjugated secondary antibody for visualization via confocal microscopy.

What criteria should be considered when selecting a CIPK9 antibody for research?

When selecting a CIPK9 antibody, consider the following criteria: (1) Specificity - the antibody should recognize CIPK9 but not other closely related CIPKs; (2) Sensitivity - it should detect physiological levels of CIPK9; (3) Species reactivity - ensure it recognizes CIPK9 from your plant species of interest; (4) Applications compatibility - verify it works in your intended applications (Western blot, immunoprecipitation, ChIP, etc.); and (5) Validation data - check for evidence of specificity testing, including use of knockout/mutant controls like the cipk9-1 and cipk9-2 mutants . Polyclonal antibodies often provide higher sensitivity but may have more cross-reactivity, while monoclonal antibodies offer higher specificity but potentially lower sensitivity.

How can I validate a CIPK9 antibody's specificity in my experimental system?

To validate CIPK9 antibody specificity:

• Genetic controls: Test the antibody on samples from wild-type plants alongside cipk9 mutants (such as cipk9-1 and cipk9-2) . The antibody should show a signal in wild-type samples but not in the mutants.

• Recombinant protein controls: Express and purify recombinant CIPK9 protein and use it as a positive control.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to your samples. This should abolish specific signals.

• Cross-reactivity testing: Test against recombinant proteins of closely related CIPKs (CIPK8, CIPK14, CIPK15, and CIPK23) to ensure specificity .

• Western blot profile: Verify that the detected band matches the expected molecular weight of CIPK9 (approximately 54 kDa).

How can CIPK9 antibodies be used to study protein-protein interactions with CBLs?

CIPK9 antibodies can be valuable tools for studying protein-protein interactions through several methodological approaches:

• Co-immunoprecipitation (Co-IP): Use anti-CIPK9 antibodies to pull down CIPK9 from plant extracts, followed by immunoblotting for potential interacting partners like CBL2 or CBL3. Research has shown that CIPK9 interacts with seven CBLs (CBL1-3, CBL5, CBL6, CBL8, and CBL9), with the strongest interactions observed with CBL2 and CBL3 .

• Reciprocal pull-down assays: Similar to what has been demonstrated in the literature, use GST-tagged CBL proteins to pull down CIPK9 from plant extracts expressing cMyc-CIPK9, then detect using anti-cMyc or anti-CIPK9 antibodies .

• Proximity ligation assay (PLA): This technique can visualize protein interactions in situ by generating fluorescent signals when two proteins are in close proximity (<40 nm).

• ChIP-reChIP: For studying interactions in chromatin contexts if CIPK9 is involved in transcriptional regulation complexes.

What are the recommended protocols for using CIPK9 antibodies in immunolocalization studies?

For successful immunolocalization of CIPK9:

• Sample preparation: Fix plant tissues in 4% paraformaldehyde in PBS for 2-4 hours, followed by dehydration and embedding in paraffin or resin.

• Sectioning: Prepare 5-10 μm sections and mount on poly-L-lysine coated slides.

• Antigen retrieval: Treat sections with citrate buffer (pH 6.0) at 95°C for 10-15 minutes to expose epitopes.

• Blocking: Block with 3% BSA in PBS containing 0.1% Triton X-100 for 1 hour at room temperature.

• Primary antibody incubation: Apply CIPK9 antibody (1:100-1:500 dilution) and incubate overnight at 4°C. For co-localization studies with CBL2 or CBL3, include antibodies against these proteins.

• Secondary antibody: Use fluorophore-conjugated secondary antibodies (1:500-1:1000) specific to the host species of your primary antibodies.

• Counterstaining: Stain nuclei with DAPI and membranes with appropriate trackers if needed.

• Visualization: Image using confocal microscopy with appropriate filter settings.

When analyzing results, compare with the reported tonoplast and plasma membrane localization patterns observed when CIPK9 interacts with CBL2 and CBL3 .

How can CIPK9 antibodies be used to investigate the phosphorylation state of CIPK9?

To investigate CIPK9 phosphorylation states:

• Phospho-specific antibodies: Use antibodies specifically designed to recognize phosphorylated CIPK9 at key residues.

• Phos-tag SDS-PAGE: Incorporate Phos-tag into polyacrylamide gels to separate phosphorylated from non-phosphorylated CIPK9, then detect with standard CIPK9 antibodies.

• Lambda phosphatase treatment: Treat samples with lambda phosphatase before immunoblotting to confirm that mobility shifts are due to phosphorylation.

• Immunoprecipitation followed by mass spectrometry: Use CIPK9 antibodies to immunoprecipitate the protein, then analyze by mass spectrometry to identify phosphorylation sites.

This approach is particularly relevant given that AP2C1 (a protein phosphatase 2C) has been shown to dephosphorylate the auto-phosphorylated form of CIPK9 in vitro, presenting a regulatory mechanism for CIPK9 function .

How can I use CIPK9 antibodies to study the regulatory interactions between CIPK9 and AP2C1?

To study CIPK9-AP2C1 interactions:

• Co-immunoprecipitation: Use anti-CIPK9 antibodies to pull down CIPK9 complexes and probe for AP2C1, or vice versa.

• In vitro phosphatase assays: Immunopurify CIPK9 using specific antibodies, allow auto-phosphorylation, then incubate with recombinant AP2C1 and monitor dephosphorylation. This approach has been used to demonstrate that AP2C1 dephosphorylates the auto-phosphorylated form of CIPK9 .

• BiFC complementation: Use split YFP fusions with CIPK9 and AP2C1 to visualize interactions in vivo, as has been done for CIPK9-CBL interactions .

• FRET analysis: Tag CIPK9 and AP2C1 with appropriate fluorophores and measure energy transfer to quantify protein-protein interactions in live cells.

• Phosphorylation site mutant analysis: Generate phosphorylation site mutants of CIPK9 and use antibodies to monitor how mutations affect interactions with AP2C1.

Research has shown that AP2C1 and CIPK9 interact to regulate K+-deficiency responses in Arabidopsis, with CIPK9 functioning as a positive regulator and AP2C1 acting as a negative regulator .

What are common challenges when using CIPK9 antibodies and how can they be addressed?

How do I interpret conflicting data between CIPK9 antibody-based detection and transcript analysis?

When faced with discrepancies between CIPK9 protein levels (detected by antibodies) and transcript levels (measured by RT-PCR or RNA-seq):

• Consider post-transcriptional regulation: CIPK9 may be regulated at the translation level or through protein stability mechanisms, independent of transcript abundance.

• Examine protein degradation pathways: Protein levels might be affected by ubiquitin-mediated degradation or other proteolytic processes.

• Analyze temporal dynamics: Transcript levels may change earlier than protein levels due to the time required for translation and protein accumulation.

• Check experimental conditions: Verify that samples for protein and RNA analyses were collected under identical conditions.

• Validate with multiple methods: Use complementary approaches such as GFP-tagged CIPK9 expression lines alongside antibody detection.

• Genetic complementation: Perform complementation experiments with cipk9 mutants to validate functional relevance, as has been done with the cipk9-1 mutant .

How can CIPK9 antibodies be incorporated into ChIP assays to study its potential role in gene regulation?

For ChIP (Chromatin Immunoprecipitation) assays using CIPK9 antibodies:

• Cross-linking: Cross-link plant tissues with 1% formaldehyde for 10-15 minutes.

• Chromatin preparation: Extract nuclei, sonicate to fragment chromatin (200-500 bp fragments), and confirm fragmentation by gel electrophoresis.

• Immunoprecipitation: Incubate chromatin with pre-cleared protein A/G beads and CIPK9 antibody. Include non-immune IgG as a negative control.

• Washing and elution: Wash beads rigorously to remove non-specific binding and elute protein-DNA complexes.

• Reverse cross-linking and DNA purification: Reverse formaldehyde cross-links and purify DNA.

• qPCR or sequencing: Analyze enriched DNA by qPCR with primers for potential target genes or by next-generation sequencing (ChIP-seq).

While there is no direct evidence of CIPK9 binding to DNA, this approach could help investigate whether CIPK9 forms part of transcriptional complexes regulating K+ deficiency responses or interacts with transcription factors like IDD10, which has been shown to directly bind to the CIPK9 promoter .

What experimental design would you recommend to investigate CIPK9's role in the cross-talk between potassium deficiency and ammonium toxicity using CIPK9 antibodies?

To investigate CIPK9's role in K+ deficiency and NH4+ toxicity cross-talk:

• Growth conditions matrix:

  • Control (sufficient K+, no NH4+)

  • Low K+ (deficient K+, no NH4+)

  • Ammonium stress (sufficient K+, high NH4+)

  • Combined stress (deficient K+, high NH4+)

• Genetic materials:

  • Wild-type plants

  • cipk9 mutants (cipk9-1 and cipk9-2)

  • idd10 mutants

  • ap2c1 mutants (ap2c1-1 and ap2c1-2)

  • CIPK9 complementation and overexpression lines

• Analytical methods:

  • Use CIPK9 antibodies for immunoblotting to quantify protein levels across conditions

  • Immunoprecipitate CIPK9 and analyze phosphorylation status

  • Perform co-IP to identify condition-specific protein interactions

  • Conduct immunolocalization to determine if CIPK9 localization changes under different stress conditions

• Physiological measurements:

  • K+ content analysis using atomic absorption spectroscopy

  • Root growth measurements

  • Chlorophyll content determination

• Gene expression analysis:

  • Monitor expression of K+-deficiency and NH4+ response genes

This experimental design would help determine if CIPK9's roles in K+ homeostasis and NH4+ sensitivity are linked, and how its protein levels, phosphorylation state, and interactions change under different nutrient stress conditions .