CKI1 Antibody

What is CKI1 and why are antibodies against it important in plant research?

CKI1 (CYTOKININ-INDEPENDENT1) is a membrane-bound sensor histidine kinase that plays crucial roles in plant development, particularly in female gametophyte formation and vascular tissue development in Arabidopsis thaliana. Antibodies against CKI1 are valuable tools in plant biology research because they allow researchers to precisely detect and study this specific histidine kinase within complex biological samples. The gene redundancy and high homology among histidine kinase family members often complicates the unambiguous identification of individual proteins, making specific antibodies particularly valuable.

CKI1 acts upstream of Histidine Phosphotransfer Proteins (AHPs) to regulate downstream signaling events in a cytokinin receptor-independent manner. This CKI1-AHP signaling pathway is essential for plant growth and development, making antibodies against CKI1 invaluable for studying these critical developmental processes.

What is the difference between plant CKI1 antibodies and mammalian CK1/CKI antibodies?

While their names are similar, plant CKI1 and mammalian CK1 (Casein Kinase 1) are distinct proteins with different functions:

• Plant CKI1: A histidine kinase involved in cytokinin signaling in plants like Arabidopsis. Antibodies against plant CKI1 typically target the receiver domain (CKI1RD) and are used to study plant development, particularly female gametogenesis and vascular tissue formation.

• Mammalian CK1/CKI-alpha/CSNK1A1: A serine/threonine protein kinase that belongs to the protein kinase superfamily. This cytoplasmic protein participates in Wnt signaling, phosphorylates multiple proteins including CTNNB1 at 'Ser-45', and may play a role in chromosome segregation during mitosis. Antibodies against mammalian CK1 are used in cancer research, particularly for non-small cell lung cancer (NSCLC) studies.

When selecting antibodies for your research, it's crucial to verify which protein the antibody targets to avoid experimental confusion and misinterpretation of results.

How can I validate the specificity of CKI1 antibodies for my research?

Validating antibody specificity is crucial for reliable experimental results. For CKI1 antibodies, employ these validation strategies:

• Comparative analysis with homologous proteins: Test the antibody against highly homologous members of the same protein family. For plant CKI1, compare with other histidine kinases like AHK4, CKI2, and ETR1. Published research demonstrates that properly generated anti-CKI1RD antibodies can specifically distinguish CKI1 from these homologous proteins both in vitro and in situ.

• Western blot with recombinant protein: Use purified recombinant CKI1 protein as a positive control. Antibodies like those targeting the receiver domain of CKI1 (CKI1RD) have demonstrated a detection limit of 10 ng at a 1:20,000 dilution.

• Pull-down assays: Verify antibody specificity by conducting pull-down assays with CKI1-GFP fusion proteins from plant cell lysates. Successful pull-down of only the target protein and not related family members confirms specificity.

• Knockout/knockdown controls: Include samples from CKI1 knockout or knockdown plants as negative controls when available. The rare viable homozygous cki1-8 mutant Arabidopsis plants could serve as valuable controls.

What are the recommended experimental conditions for using CKI1 antibodies in Western blotting?

For optimal Western blot results with CKI1 antibodies, consider these technical recommendations:

• Sample preparation:

  • For plant CKI1: Extract total protein using a buffer containing phosphatase inhibitors to preserve phosphorylation states.

  • For purified recombinant CKI1RD: Use as a positive control at 10-50 ng per lane.

• Antibody dilution:

  • Primary antibody (anti-CKI1RD): Start with 1:20,000 dilution, which has demonstrated detection limits of approximately 10 ng of purified target protein.

  • For commercial antibodies like mouse monoclonal CK1 antibodies, optimal dilutions should be determined empirically, but manufacturers often recommend starting with 1:1000 dilution.

• Detection method:

  • Enhanced chemiluminescence (ECL) systems work well for detecting CKI1 antibody binding.

  • For lower abundance CKI1 in plant samples, consider using more sensitive detection methods like ECL-Plus.

• Expected molecular weight:

  • For plant CKI1: Verify bands at the expected molecular weight.

  • For mammalian CK1/CSNK1A1: Look for bands around 39 kDa.

What techniques can I use to isolate and purify CKI1 protein for antibody production?

To produce high-quality antibodies against CKI1, you need properly purified protein. Here's a methodological approach based on published protocols:

• Domain selection: The receiver domain (RD) of CKI1 has proven effective as an antigen. This C-terminal domain can be specifically expressed in bacterial systems to obtain a unique antigen that minimizes cross-reactivity with other histidine kinases.

• Expression system:

  • Use IPTG-inducible expression systems in E. coli for the CKI1 receiver domain.

  • For full-length CKI1, consider baculovirus-insect cell expression systems which maintain proper protein folding and post-translational modifications.

• Purification protocols:

  • For CKI1RD: Express with affinity tags (like GST or His) for easier purification.

  • Use column chromatography followed by additional purification steps to achieve >90% purity.

  • Verify purity by SDS-PAGE and consider mass spectrometry for confirmation.

• Quality control:

  • Assess protein activity if possible (for enzyme domains).

  • Check for endotoxin levels (should be <1.0 EU per μg).

  • Verify protein integrity and proper folding through circular dichroism or tryptophan fluorescence if specialized equipment is available.

How can I use CKI1 antibodies to study protein-protein interactions in cytokinin signaling pathways?

CKI1 antibodies are powerful tools for investigating protein-protein interactions in cytokinin signaling pathways. Consider these advanced methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-CKI1 antibodies to pull down CKI1 along with its interacting partners from plant lysates.

  • Analyze the precipitated proteins by mass spectrometry to identify novel interaction partners.

  • This approach has successfully demonstrated interactions between CKI1 and AHP proteins.

• Proximity labeling coupled with immunoprecipitation:

  • Express CKI1 fused to promiscuous biotin ligases (BioID or TurboID).

  • Use anti-CKI1 antibodies to verify the expression and localization of the fusion protein.

  • Proximity-labeled proteins can be purified using streptavidin and identified using mass spectrometry.

• Förster Resonance Energy Transfer (FRET) combined with immunolocalization:

  • Use antibodies to verify the expression and localization of CKI1 and potential interacting partners.

  • Perform FRET experiments to determine if the proteins are in close proximity in vivo.

  • Confirm interactions with complementary techniques like split-GFP or BiFC.

• Chromatin immunoprecipitation (ChIP) for transcription factors downstream of CKI1:

  • Use anti-CKI1 antibodies to examine how CKI1 affects the binding of downstream transcription factors to gene promoters.

  • Combine with transcriptome analyses to correlate protein interactions with gene expression changes.

What are the best approaches for in situ immunolocalization of CKI1 in plant tissues?

In situ immunolocalization of CKI1 in plant tissues requires careful methodological considerations to preserve antigenicity while maintaining tissue structure:

• Tissue fixation and embedding:

  • For paraffin sections: Fix tissues in 4% paraformaldehyde or FAA (Formalin-Acetic acid-Alcohol).

  • For cryosections: Flash freeze in OCT compound after minimal fixation to preserve antigenicity.

  • Consider alternative embedding media like LR White resin for better preservation of membrane proteins like CKI1.

• Antigen retrieval:

  • Perform citrate buffer (pH 6.0) heat-induced epitope retrieval to expose masked epitopes.

  • Optimize retrieval conditions empirically, as over-retrieval can damage tissue morphology.

• Antibody incubation:

  • Block with appropriate serum (5-10%) to reduce background.

  • Use anti-CKI1RD antibodies at optimized dilutions (start with 1:100-1:500).

  • Include controls: primary antibody omission, pre-immune serum, and competitive inhibition with purified antigen.

• Detection methods:

  • For brightfield microscopy: Use HRP-conjugated secondary antibodies with DAB substrate.

  • For fluorescence: Use fluorophore-conjugated secondary antibodies compatible with your microscopy setup.

  • Include DAPI or propidium iodide counterstaining for nuclear/cell wall visualization.

• Validation of specificity:

  • Use tissues from cki1-8 mutants as negative controls when possible.

  • Perform parallel immunolocalizations with antibodies against known markers of relevant cell types or structures.

How can CKI1 antibodies help distinguish between CKI1-dependent and cytokinin receptor-dependent signaling pathways?

Distinguishing between CKI1-dependent and canonical cytokinin receptor-dependent signaling is crucial for understanding plant development. Here's how CKI1 antibodies can facilitate this differentiation:

• Comparative immunoprecipitation:

  • Use anti-CKI1 antibodies to immunoprecipitate CKI1 complexes from wild-type plants and cytokinin receptor mutants.

  • Identify proteins that associate with CKI1 regardless of cytokinin receptor status.

  • This approach can reveal CKI1-specific signaling components that function independently of canonical cytokinin perception.

• Phosphorylation state analysis:

  • Develop phospho-specific antibodies against key phosphorylation sites in CKI1 and downstream components.

  • Compare phosphorylation patterns in response to cytokinin treatment versus CKI1 overexpression.

  • Use Western blotting with phospho-specific and total CKI1 antibodies to determine activation states.

• Temporal dynamics studies:

  • Use CKI1 antibodies in time-course experiments following cytokinin treatment.

  • Compare with constitutive CKI1 activation systems to determine differences in signaling kinetics.

  • Apply these antibodies in co-immunoprecipitation at different time points to capture dynamic protein interactions.

• Spatial localization comparisons:

  • Perform double immunolocalization with anti-CKI1 and anti-cytokinin receptor antibodies.

  • Identify tissues where CKI1 is expressed without cytokinin receptors, suggesting independent functions.

  • Correlate protein localization with known developmental phenotypes from genetic studies.

What are common issues with CKI1 antibodies in immunoprecipitation and how can I resolve them?

Immunoprecipitation with CKI1 antibodies can present several challenges. Here are common issues and their solutions:

• Poor or no pull-down of CKI1 protein:

  • Cause: Epitope masking in native protein complexes.

  • Solution: Use mild detergents (0.5-1% NP-40 or Triton X-100) in lysis buffers. Consider changing the antibody to one targeting a different epitope of CKI1.

  • Evidence-based approach: Anti-CKI1RD antibodies have demonstrated success in pulling down CKI1-GFP fusion protein from Arabidopsis cell suspension cultures, confirming their effectiveness for immunoprecipitation.

• High background or non-specific binding:

  • Cause: Insufficient blocking or washing.

  • Solution: Pre-clear lysates with Protein A/G beads, increase blocking agent concentration (BSA or non-fat milk), and perform more stringent washes with higher salt concentrations.

  • Advanced option: Consider crosslinking the antibody to beads to eliminate antibody contamination in the eluted sample.

• Degradation of CKI1 during immunoprecipitation:

  • Cause: Protease activity during lengthy procedures.

  • Solution: Use comprehensive protease inhibitor cocktails, keep samples cold, and minimize procedure time.

  • Additional approach: Consider membrane protein-specific extraction methods to better preserve membrane-bound CKI1.

• Co-precipitating proteins mask CKI1 detection:

  • Cause: Strong interactions with other proteins.

  • Solution: Use two-step immunoprecipitation or include mild denaturants like 0.1% SDS in the wash buffer.

  • Analysis strategy: Consider mass spectrometry rather than Western blotting for complex samples.

How should I modify protocols when using CKI1 antibodies for different plant species?

When adapting CKI1 antibody-based protocols across plant species, consider these methodological adjustments:

• Homology assessment and antibody selection:

  • Perform sequence alignments of CKI1 homologs across target species.

  • Select antibodies targeting highly conserved regions, such as the receiver domain, which tends to be more conserved than other domains.

  • For divergent species, consider raising new antibodies against species-specific peptides.

• Extraction buffer modifications:

  • Adjust extraction buffers based on species-specific tissue composition:

    • For species with high phenolic content: Add PVPP (polyvinylpolypyrrolidone) and β-mercaptoethanol.

    • For species with high proteolytic activity: Increase protease inhibitor concentrations.

• Immunodetection protocol adjustments:

  • Western blotting: Start with a lower dilution of antibody (1:1000 instead of 1:20,000) for new species.

  • Immunolocalization: Increase antibody concentration and incubation time for tissues with dense cell walls.

  • For recalcitrant tissues: Consider extended antigen retrieval treatments.

• Validation strategies:

  • Use recombinant protein from the target species as a positive control.

  • Include appropriate negative controls (pre-immune serum, antibody blocking with antigen).

  • When possible, validate with genetic knockouts or knockdowns in the target species.

What are the best approaches for multiplexing CKI1 antibodies with other antibodies in co-localization studies?

Multiplexing CKI1 antibodies with other antibodies can provide valuable insights into protein co-localization and interaction dynamics. Here are methodological recommendations:

• Antibody compatibility planning:

  • Host species selection: Choose primary antibodies raised in different host species (e.g., rabbit anti-CKI1 with mouse anti-AHP).

  • Isotype consideration: If antibodies are from the same host, use different isotypes (IgG1, IgG2a, etc.) and isotype-specific secondary antibodies.

  • Validation: Test each antibody individually before multiplexing to establish baseline signals and specificity.

• Sequential immunostaining protocol:

  • For challenging combinations: Apply primary antibodies sequentially with an elution/blocking step between.

  • Method: Apply first primary antibody, detect with first secondary antibody, capture images, then elute antibodies using glycine buffer (pH 2.5) or SDS buffer, block again, and apply the second primary antibody.

  • Advantages: Eliminates cross-reactivity concerns but requires stable samples that can withstand multiple treatments.

• Fluorophore selection and detection optimization:

  • Spectrum separation: Choose fluorophores with minimal spectral overlap (e.g., Alexa 488 and Alexa 647).

  • Signal balancing: Adjust antibody concentrations to achieve comparable signal intensities.

  • Controls: Include single-antibody controls on the same slide/sample to confirm absence of bleed-through.

• Advanced imaging approaches:

  • Sequential scanning: Use sequential rather than simultaneous scanning in confocal microscopy.

  • Spectral unmixing: Apply spectral unmixing algorithms for fluorophores with partial overlap.

  • Super-resolution techniques: Consider STED or SIM microscopy for detailed co-localization analyses of CKI1 with interacting partners.

How might CKI1 antibodies facilitate research into the evolutionary conservation of cytokinin signaling pathways?

CKI1 antibodies can serve as powerful tools for exploring the evolutionary conservation of cytokinin signaling across plant species and throughout evolutionary history:

• Comparative immunodetection across species:

  • Use anti-CKI1RD antibodies to detect homologous proteins across diverse plant lineages.

  • Create an immunological profile of CKI1-like proteins from bryophytes to angiosperms.

  • Correlate protein presence, abundance, and localization with the complexity of reproductive structures across evolutionary time.

• Analysis of functional domain conservation:

  • Develop domain-specific antibodies targeting different regions of CKI1.

  • Use these antibody panels to assess which domains are most conserved across evolutionary distance.

  • Combine with functional assays to determine if conserved domains maintain similar activities.

• Co-evolution of signaling components:

  • Apply CKI1 antibodies alongside antibodies against AHPs and other downstream components.

  • Investigate whether CKI1-AHP interactions are conserved across species with different developmental complexities.

  • Use co-immunoprecipitation to identify lineage-specific interaction partners that may reveal evolutionary adaptations.

• Ancestral state reconstruction approaches:

  • Use immunological data to supplement sequence-based phylogenetic analyses.

  • Develop hypotheses about the ancestral functions of CKI1-like proteins in early land plants.

  • Test these hypotheses through heterologous expression of ancestral sequence reconstructions and antibody-based detection.

What potential exists for developing monoclonal antibodies against specific phosphorylated forms of CKI1?

Developing phospho-specific monoclonal antibodies against CKI1 would significantly advance our understanding of histidine kinase signaling dynamics in plants:

• Target phosphorylation site identification:

  • Conduct mass spectrometry analysis to identify specific phosphorylation sites on CKI1 under different conditions.

  • Focus on conserved histidine residues in the kinase domain and aspartate residues in the receiver domain.

  • Prioritize sites that show dynamic phosphorylation in response to developmental cues or environmental signals.

• Peptide design considerations:

  • Design phosphopeptides (10-15 amino acids) with the phosphorylated residue centrally located.

  • Include carrier proteins like KLH for immunization.

  • Consider dual-phosphorylation state antibodies if multiple phosphorylation events occur in close proximity.

• Validation methods for phospho-specific antibodies:

  • Test against phosphorylated and non-phosphorylated recombinant proteins.

  • Use phosphatase treatments as negative controls.

  • Verify specificity using phosphomimetic mutants (e.g., His→Glu) and non-phosphorylatable mutants (e.g., His→Ala).

• Potential applications:

  • Tracking phosphorylation dynamics during plant development.

  • Monitoring CKI1 activation states in response to various stimuli.

  • Comparing phosphorylation patterns between wild-type and mutant plants to understand regulatory mechanisms.

How can CKI1 antibodies contribute to understanding the role of this protein in plant stress responses?

While CKI1 is primarily studied in developmental contexts, emerging evidence suggests potential roles in stress responses. CKI1 antibodies can help elucidate these functions:

• Stress-induced expression and localization changes:

  • Use immunoblotting and immunolocalization to monitor CKI1 protein levels and subcellular distribution under various stresses (drought, salinity, pathogen attack).

  • Compare with transcript-level changes to identify post-transcriptional regulation.

  • Correlate protein levels with physiological responses to identify potential causal relationships.

• Stress-specific protein interaction networks:

  • Apply immunoprecipitation with anti-CKI1RD antibodies under normal and stress conditions.

  • Identify stress-specific interaction partners through mass spectrometry.

  • Validate key interactions with targeted co-immunoprecipitation experiments.

• Cross-talk with established stress signaling pathways:

  • Investigate potential interactions between CKI1 and components of ABA, ethylene, or jasmonate signaling.

  • Use double immunolabeling to identify tissues where CKI1 and stress hormone receptors co-localize.

  • Apply CKI1 antibodies in plants with mutations in stress signaling components to establish epistatic relationships.

• Potential applications in crop improvement:

  • Use CKI1 antibodies to screen for naturally occurring variants with altered expression or stability.

  • Correlate protein characteristics with stress tolerance phenotypes.

  • Develop diagnostic tools to predict stress responses based on CKI1 protein profiles in crop species.

What quality control parameters should I assess when selecting or validating CKI1 antibodies?

When selecting or validating CKI1 antibodies, assess these critical quality control parameters:

What are the recommended storage and handling conditions for maintaining CKI1 antibody activity?

Proper storage and handling of CKI1 antibodies is essential for maintaining their activity and extending their usable lifespan:

• Storage temperature:

  • Store antibody stock solutions at -20°C for long-term storage.

  • For working aliquots, store at 4°C with preservatives for up to 2 weeks.

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots (typically 10-20 μL).

• Buffer composition considerations:

  • Optimal pH: Maintain pH between 6.5-8.0 for most applications.

  • Preservatives: Include 0.02% sodium azide to prevent microbial growth in working solutions.

  • Stabilizers: Consider adding 50% glycerol for freezing stability.

  • Carrier proteins: For dilute solutions (<0.1 mg/mL), add BSA (1-5 mg/mL) to prevent adsorption to container surfaces.

• Handling practices:

  • Always wear gloves to prevent contamination with skin proteins and proteases.

  • Use sterile pipette tips and containers.

  • Centrifuge antibody solutions briefly before opening to collect liquid at the bottom of the tube.

  • Allow refrigerated antibodies to equilibrate to room temperature before opening to prevent condensation.

• Stability monitoring:

  • Periodically test activity against a standard sample.

  • Document performance over time to establish realistic expiration dates.

  • Consider including positive controls in each experiment to verify antibody performance.

• Shipping conditions:

  • For CKI1 antibody products, shipping should occur at frozen temperature with blue ice/gel packs.

  • Upon receipt, store immediately at -20°C to maintain stability for up to 6 months.

What is the molecular structure and key domains of CKI1 protein?

Understanding the molecular structure of CKI1 is essential for designing effective antibody-based experiments:

Plant CKI1 (Arabidopsis thaliana):

• A membrane-bound histidine kinase with a multi-domain structure

• Contains a cytokinin-binding CHASE domain (though CKI1 activation is cytokinin-independent)

• Features a histidine kinase domain with the conserved His residue that becomes phosphorylated

• Includes a C-terminal receiver domain (CKI1RD) with a conserved Asp residue that accepts the phosphoryl group from the histidine residue

Mammalian CK1/CSNK1A1:

• A cytoplasmic serine/threonine protein kinase

• Belongs to the protein kinase superfamily, CK1 Ser/Thr protein kinase family

• Full amino acid sequence: MASSSGSKAEFIVGGKYKLVRKIGSGSFGDIYLAINITNGEEVAVKLESQKARHPQLLYESKLYKILQGGVGIPHIRWYGQEKDYNVLVMDLLGPSLEDLFNFCSRRFTMKTVLMLADQMISRIEYVHTKNFIHRDIKPDNFLMGIGRHCNKLFLIDFGLAKKYRDNRTRQHIPYREDKNLTGTARYASINAHLGIEQSRRDDMESLGYVLMYFNRTSLPWQGLKAATKKQKYEKISEKKMSTPVEVLCKGFPAEFAMYLNYCRGLRFEEAPDYMYLRQLFRILFRTLNHQYDYTFDWTMLKQKAAQQAASSSGQGQQAQTPTGKQTDKTKSNMKGF

• Molecular weight: 39 kDa (calculated)

What post-translational modifications occur on CKI1 and how do they affect antibody recognition?

Post-translational modifications (PTMs) on CKI1 can significantly impact antibody recognition and should be considered when designing experiments:

For mammalian CK1/CSNK1A1, numerous PTMs have been documented:

For plant CKI1, the primary functional PTMs include:

• Histidine phosphorylation in the kinase domain

• Aspartate phosphorylation in the receiver domain

Implications for antibody recognition:

• Epitope masking: PTMs can alter protein conformation, potentially masking epitopes recognized by antibodies.

• Modification-specific recognition: Some antibodies may preferentially bind to modified or unmodified forms of the protein.

• Experimental considerations:

  • Include phosphatase inhibitors in extraction buffers when studying phosphorylated forms.

  • Consider using phosphatase treatment as a control to verify phosphorylation-dependent recognition.

  • For ubiquitination studies, include deubiquitinase inhibitors.

How does the expression profile of CKI1 vary across different plant tissues and developmental stages?

Understanding the tissue-specific and developmental expression patterns of CKI1 is crucial for experimental design and interpretation of antibody-based results:

• Female gametophyte expression:

  • CKI1 is essential for female gametogenesis in Arabidopsis.

  • Mutations in CKI1 typically cause female gametophytic lethality, though the cki1-8 allele shows reduced transmission through female gametophytes (~0.17%).

  • Homozygous cki1-8 mutant plants exhibit defective megagametogenesis, confirming the critical role of CKI1 in this developmental process.

• Vascular tissue expression:

  • CKI1 regulates vascular tissue development in Arabidopsis.

  • Anti-CKI1 immunolocalization can be used to examine protein distribution in vascular tissues at different developmental stages.

• Developmental dynamics:

  • CKI1 protein levels may vary throughout plant development.

  • Homozygous cki1-8 mutant plants grow larger than wild-type plants and rarely set enlarged seeds, suggesting roles beyond gametogenesis.

• Experimental applications:

  • Use immunohistochemistry with anti-CKI1RD antibodies to create a detailed expression atlas across tissues and developmental stages.

  • Combine with in situ hybridization to correlate protein and mRNA localization.

  • Compare CKI1 protein distribution with known cytokinin response markers to identify potential sites of CKI1-specific signaling.