CKX5 Antibody

What is CKX5 and why is it significant in developing targeted antibodies?

CKX5 (Cytokinin Oxidase/Dehydrogenase 5) is an enzyme involved in cytokinin signaling and biosynthesis in plants, particularly in Arabidopsis. It has gained significant research interest due to its role in plant immunity against pathogens like Botrytis cinerea. Studies have demonstrated that CKX5 expression is significantly induced in B. cinerea-infected leaves and subsequently in distant untreated leaves of the same plant, suggesting its involvement in systemic acquired resistance .

Developing antibodies against CKX5 is valuable for multiple reasons:

• Enables protein-level verification of gene expression changes observed in transcriptional studies

• Allows for subcellular localization studies to understand CKX5's spatial distribution during pathogen responses

• Facilitates investigation of post-translational modifications that may regulate CKX5 activity

• Provides tools for studying protein-protein interactions involving CKX5 in immune signaling pathways

How do CKX5 antibodies differ from antibodies against other CKX family members?

When developing or selecting antibodies against CKX5, researchers must consider several factors to ensure specificity against other CKX family proteins:

Antibody development should target unique epitopes in the CKX5 sequence that differ from other family members, particularly focusing on variable regions outside the conserved catalytic domains . Cross-reactivity testing against other CKX family proteins is essential for validation, similar to approaches used for discriminating between related proteins like cytokeratins .

What techniques are most effective for validating CKX5 antibody specificity?

For rigorous validation of CKX5 antibody specificity, researchers should implement a multi-tiered approach:

• Western blot analysis with recombinant proteins:

  • Test against purified recombinant CKX5 protein

  • Include other recombinant CKX family members as negative controls

  • Verify expected molecular weight and single band detection

• Genetic validation approaches:

  • Compare antibody signals between wild-type plants and CKX5 knockout/overexpression lines

  • The antibody signal should be absent in knockout lines and enhanced in overexpression lines

• Immunoprecipitation followed by mass spectrometry:

  • Confirms that the antibody is pulling down CKX5 and not other proteins

  • Identifies any cross-reactive proteins for further validation

• Kinetic analysis using flow cytometry:

  • Determines binding affinity (KD) to ensure high specificity

  • Similar to methods used for CXCR5 antibody validation that achieved dissociation constants of 7.2 × 10⁻¹⁰ M

• Competitive binding assays:

  • Pre-absorption of the antibody with purified antigen should eliminate signal

  • Helps distinguish specific from non-specific binding

How should experiments be designed to study CKX5 protein dynamics during pathogen infection?

When designing experiments to study CKX5 protein dynamics during pathogen infection using antibodies, researchers should consider:

Experimental timeline and sampling:

• Collect samples at multiple timepoints (14h, 24h, 48h post-infection) to capture the dynamic expression pattern of CKX5, reflecting the transcriptional changes observed in qRT-PCR studies

• Include both infected and distant uninfected tissues to investigate systemic responses

Controls and comparisons:

• Wild-type plants vs. CKX5-overexpressing plants (showed enhanced resistance to B. cinerea)

• ERF6 and AHL15 mutant plants (these transcription factors affect CKX5 regulation)

• Mock-inoculated plants as negative controls

Quantification methods:

• Standardize protein extraction procedures for consistent yield

• Use internal loading controls (constitutively expressed proteins) for normalization

• Implement densitometric analysis for Western blots with statistical validation

• Consider flow cytometry for single-cell level quantification of protein expression

Tissue-specific considerations:

• Compare CKX5 protein levels in different plant tissues

• Correlate with CKX5:GUS reporter analysis to validate tissue-specific expression patterns

What methodological approaches can be used to study the relationship between CKX5 and transcription factors?

Given that transcription factors including WRKY40, WRKY33, ERF6, AHL15, AHL17, ANAC003, TCP13, and ANAC019 are induced similarly to CKX5 during pathogen infection , several methodological approaches using CKX5 antibodies can illuminate these relationships:

• Chromatin Immunoprecipitation (ChIP) followed by qPCR:

  • Use antibodies against the transcription factors to precipitate protein-DNA complexes

  • Quantify enrichment of CKX5 promoter sequences in precipitated material

  • Validate yeast one-hybrid findings in planta

• Co-immunoprecipitation (Co-IP) assays:

  • Use CKX5 antibodies to precipitate the protein and associated complexes

  • Probe for co-precipitated transcription factors

  • Determine if protein-protein interactions occur beyond DNA binding

• Immunofluorescence co-localization:

  • Double-label experiments with CKX5 antibody and antibodies against transcription factors

  • Determine if co-localization occurs during pathogen response

  • Track temporal changes in subcellular localization

• Protein expression analysis in transcription factor mutants:

  • Compare CKX5 protein levels in ERF6-overexpressing plants and ERF6/AHL15-knockout mutants

  • Correlate protein changes with observed transcript level alterations

  • Establish causality in the regulatory relationship

• Proximity ligation assay:

  • Detect direct protein-protein interactions between CKX5 and transcription factors at endogenous levels

  • Visualize interaction events in situ within plant cells

How can researchers develop high-affinity monoclonal antibodies against CKX5?

Developing high-affinity monoclonal antibodies against CKX5 requires strategic planning and methodological rigor:

• Antigen design and preparation:

  • Express full-length CKX5 or specific peptide regions in heterologous systems

  • Target unique epitopes by analyzing sequence alignments of CKX family proteins

  • Ensure proper protein folding for conformational epitopes or use synthetic peptides for linear epitopes

• Immunization and hybridoma generation:

  • Immunize animals (typically rats or mice) with purified CKX5 protein

  • Harvest B cells and fuse with myeloma cells to generate hybridomas

  • Screen hybridoma supernatants for CKX5-specific antibodies

• Comprehensive screening strategy:

  • Primary screening by ELISA against purified CKX5

  • Secondary screening by Western blot and flow cytometry using CKX5-overexpressing cells

  • Counter-screening against other CKX family proteins to confirm specificity

• Kinetic analysis optimization:

  • Measure dissociation constants (KD) using flow cytometry, aiming for high-affinity binding in the nanomolar to picomolar range

  • Similar to methods used for other receptor antibodies that achieved KD values of 7.2 × 10⁻¹⁰ M

• Clone selection and antibody production:

  • Select stable hybridoma clones showing highest affinity and specificity

  • Expand selected clones and purify antibodies using protein A/G chromatography

  • Validate purified antibodies through multiple assays before experimental use

• Isotype determination:

  • Determine antibody isotype (e.g., IgG2b, kappa) for optimal application selection

  • Different isotypes have varying properties for immunoprecipitation, Western blotting, and immunohistochemistry

How should researchers interpret discrepancies between CKX5 gene expression and protein levels?

When facing discrepancies between CKX5 transcript abundance and protein levels detected by antibodies, researchers should consider:

• Post-transcriptional regulation mechanisms:

  • microRNA-mediated repression of translation

  • mRNA sequestration or storage

  • Altered mRNA stability

• Post-translational regulation:

  • Protein degradation rates may vary during stress responses

  • Ubiquitin-mediated proteolysis may target CKX5 during specific phases

  • Post-translational modifications may mask antibody epitopes

• Temporal considerations:

  • Implement time-course studies covering hours to days post-infection

  • Protein accumulation typically lags behind transcript induction

  • Compare the 14h, 24h, and 48h timepoints observed in transcript studies

• Methodological approach:

  • Use multiple antibodies targeting different CKX5 epitopes

  • Combine Western blot with immunohistochemistry for spatial information

  • Implement pulse-chase experiments to determine protein turnover rates

• Data integration strategies:

  • Create mathematical models accounting for transcription, translation, and degradation rates

  • Normalize protein data to account for extraction efficiency variations

  • Consider relative changes rather than absolute values when comparing transcript and protein data

What statistical approaches are most appropriate for quantifying CKX5 immunostaining patterns?

For rigorous quantification of CKX5 immunostaining patterns, researchers should employ:

• Image acquisition standardization:

  • Fixed exposure times and microscope settings across all samples

  • Multiple fields per sample to account for tissue heterogeneity

  • Z-stack imaging for three-dimensional analysis when appropriate

• Quantification methods:

  • Mean fluorescence intensity measurements in defined regions of interest

  • Colocalization analysis with subcellular markers (Pearson's or Mander's coefficients)

  • Cell counting for determining percentage of CKX5-positive cells

• Statistical analysis approaches:

  • For comparing two conditions (e.g., infected vs. uninfected): paired t-tests or Wilcoxon signed-rank tests

  • For multiple conditions/timepoints: ANOVA with appropriate post-hoc tests

  • For correlation analysis: Pearson's or Spearman's correlation coefficients

• Data visualization:

  • Box plots showing distribution of staining intensity

  • Heat maps for spatial distribution patterns

  • Time-course graphs for temporal changes in expression

• Controls for quantification:

  • Include isotype control antibodies for background determination

  • Use CKX5 knockout tissues as negative controls

  • Include CKX5-overexpressing tissues as positive controls

How can researchers determine if antibody binding is affected by post-translational modifications of CKX5?

Determining whether antibody binding is affected by post-translational modifications (PTMs) of CKX5 requires systematic investigation:

• Epitope mapping:

  • Identify the exact binding region of the antibody on CKX5

  • Analyze whether this region contains potential PTM sites (phosphorylation, glycosylation, etc.)

  • Similar to approaches used for mapping monoclonal antibody binding sites on proteins like creatine kinase

• Enzymatic treatment experiments:

  • Treat protein samples with phosphatases, glycosidases, or other PTM-removing enzymes

  • Compare antibody binding before and after treatment

  • Enhanced signal after treatment suggests PTM interference with antibody binding

• Mass spectrometry analysis:

  • Identify specific PTMs present on CKX5 during pathogen response

  • Create a PTM profile map of CKX5 at different timepoints after infection

  • Correlate PTM changes with antibody binding efficiency

• Site-directed mutagenesis approach:

  • Generate recombinant CKX5 variants with mutations at potential PTM sites

  • Compare antibody binding to wild-type and mutant proteins

  • Reduced binding to wild-type but not mutant protein suggests PTM interference

• Differential extraction protocols:

  • Use extraction buffers that preserve or remove specific PTMs

  • Compare antibody detection efficiency under different extraction conditions

  • Develop modified Western blot protocols optimized for modified CKX5 detection

What are the optimal conditions for using CKX5 antibodies in Western blot analysis of plant tissues?

Optimizing Western blot conditions for CKX5 antibody detection in plant tissues requires attention to several factors:

• Sample preparation:

  • Extraction buffer: Use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail

  • Include reducing agents like 5 mM DTT to maintain protein structure

  • Consider plant-specific extraction additives (PVP or PVPP) to remove phenolic compounds

• Gel electrophoresis parameters:

  • Protein loading: 15-30 μg total protein per lane

  • Gel percentage: 10-12% SDS-PAGE for optimal resolution

  • Running conditions: 100V constant voltage

• Transfer optimization:

  • Membrane type: PVDF membranes typically provide better results than nitrocellulose for plant proteins

  • Transfer buffer: Standard Towbin buffer with 20% methanol

  • Transfer conditions: 100V for 1 hour or 30V overnight at 4°C

• Blocking and antibody incubation:

  • Blocking solution: 5% non-fat dry milk in TBST (preferred over BSA for plant samples)

  • Primary antibody dilution: Start with 1:1000 and optimize based on signal-to-noise ratio

  • Incubation conditions: Overnight at 4°C with gentle agitation

• Detection system optimization:

  • HRP-conjugated secondary antibodies (1:5000 dilution)

  • Enhanced chemiluminescence detection

  • Consider fluorescent secondary antibodies for multiplex detection

How can immunohistochemistry protocols be optimized for detecting CKX5 in plant tissues?

Optimizing immunohistochemistry for CKX5 detection in plant tissues requires consideration of plant-specific challenges:

• Tissue fixation and processing:

  • Fixative selection: 4% paraformaldehyde for 2-4 hours preserves protein epitopes

  • Embedding medium: Paraffin for thin sectioning or frozen sectioning for labile epitopes

  • Section thickness: 5-10 μm for optimal antibody penetration

• Antigen retrieval methods:

  • Heat-induced epitope retrieval: Citrate buffer (pH 6.0) at 95°C for 20 minutes

  • Enzymatic retrieval: Proteinase K (1-5 μg/ml) for 10 minutes at room temperature

  • Test both methods to determine optimal approach for CKX5 detection

• Blocking and permeabilization:

  • Blocking solution: 5% normal serum from secondary antibody host species plus 0.3% Triton X-100

  • Permeabilization time: 15-30 minutes for adequate antibody access

  • Include endogenous peroxidase blocking if using HRP detection systems

• Antibody incubation parameters:

  • Primary antibody dilution: 1:50 to 1:200 range (optimize empirically)

  • Incubation time: Overnight at 4°C or 2 hours at room temperature

  • Washing buffer: PBS with 0.1% Tween-20, minimum three 5-minute washes

• Detection system considerations:

  • Fluorescent detection: Alexa Fluor-conjugated secondary antibodies

  • Chromogenic detection: DAB substrate for permanent preparations

  • Signal amplification: Tyramide signal amplification for low abundance proteins

• Controls:

  • Positive control: CKX5-overexpressing plant tissues

  • Negative control: CKX5 knockout tissues and primary antibody omission

  • Competing peptide control: Pre-absorption of antibody with immunizing antigen

What approaches can optimize CKX5 antibody-based flow cytometry for plant cell analysis?

Optimizing flow cytometry for CKX5 detection in plant cells requires specialized protocols:

• Cell preparation:

  • Enzymatic digestion: Optimize cellulase/macerozyme concentrations and incubation times

  • Filtration: Use 40-70 μm mesh filters to remove cell clumps

  • Viability assessment: Include propidium iodide to exclude dead cells

• Fixation and permeabilization:

  • Fix cells with 2-4% paraformaldehyde for 15-30 minutes

  • Permeabilize with 0.1-0.3% saponin or 0.1% Triton X-100

  • Maintain buffer pH between 7.2-7.4 for optimal antibody binding

• Antibody staining protocol:

  • Primary antibody concentration: Titrate to determine optimal signal-to-noise ratio

  • Incubation time: 45-60 minutes at room temperature or 2-4 hours at 4°C

  • Secondary antibody selection: Bright fluorophores (Alexa Fluor 488 or PE) for plant cell autofluorescence compensation

• Instrument setup and analysis:

  • Autofluorescence control: Unstained cells to set baseline parameters

  • Compensation controls: Single-stained samples for each fluorophore

  • Analysis gates: Forward/side scatter to identify intact cells, followed by fluorescence gating

• Kinetic analysis optimization:

  • For determining antibody affinity (KD), use titration of antibody concentrations

  • Plot mean fluorescence intensity against antibody concentration

  • Calculate dissociation constants using non-linear regression, aiming for nanomolar to picomolar range similar to other receptor antibodies (7.2 × 10⁻¹⁰ M)

What are common issues when using CKX5 antibodies in plant research and how can they be resolved?

How can researchers address epitope masking issues in CKX5 antibody applications?

Epitope masking can significantly impact CKX5 antibody detection. Addressing this issue requires:

• Identify potential causes of epitope masking:

  • Protein-protein interactions obscuring the epitope

  • Post-translational modifications at or near the epitope

  • Conformational changes during pathogen response

  • Fixation artifacts in immunohistochemistry

• Implement multiple epitope retrieval strategies:

  • Heat-induced epitope retrieval: Test different buffers (citrate, EDTA, Tris) and pH values

  • Enzymatic digestion: Optimize proteinase K, trypsin, or pepsin concentrations and incubation times

  • Denaturants: Use urea or guanidine HCl to expose hidden epitopes

• Modify protein extraction methods:

  • Test different detergent types and concentrations

  • Include reducing agents like DTT or β-mercaptoethanol

  • Use chaotropic agents to disrupt protein-protein interactions

• Develop epitope-specific antibody panels:

  • Generate antibodies against multiple distinct regions of CKX5

  • Compare detection efficiency across different experimental conditions

  • Similar to approaches used for distinguishing between related proteins like cytokeratins

• Consider alternative detection approaches:

  • Use epitope tags in recombinant CKX5 constructs

  • Implement proximity ligation assays for enhanced sensitivity

  • Develop sandwich ELISA approaches using antibody pairs

What strategies can enhance detection sensitivity for low-abundance CKX5 protein?

For enhancing detection of low-abundance CKX5 protein, researchers should consider:

• Sample enrichment techniques:

  • Immunoprecipitation to concentrate CKX5 before analysis

  • Subcellular fractionation to isolate compartments with higher CKX5 concentration

  • Selective precipitation of protein classes

• Signal amplification methods:

  • Tyramide signal amplification (TSA) for immunohistochemistry

  • Poly-HRP secondary antibodies for Western blot

  • Biotin-streptavidin amplification systems

• Enhanced detection systems:

  • Chemiluminescence substrates with extended signal duration

  • Highly sensitive fluorophores with low background

  • Quantum dots for improved signal-to-noise ratio

• Instrument optimization:

  • Extended exposure times for Western blot imaging

  • Confocal microscopy with photomultiplier gain adjustment

  • Flow cytometry with optimized voltage settings

• Protocol modifications:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Reduce washing stringency while maintaining specificity

  • Use carrier proteins to prevent antibody adsorption to tubes

• Consider mass spectrometry-based approaches:

  • Targeted MS/MS for specific CKX5 peptides

  • Selected reaction monitoring (SRM) for quantitative analysis

  • Parallel reaction monitoring (PRM) for enhanced specificity

By implementing these strategies and understanding the specific challenges associated with CKX5 antibody research, investigators can develop robust protocols for studying this important protein in plant immunity and cytokinin signaling pathways.