CKX6 Antibody

What is CKX6 and why is it significant in plant research?

CKX6 (Cytokinin Oxidase/Dehydrogenase 6) is an enzyme involved in cytokinin degradation in plants. It plays a crucial role in regulating cytokinin levels, which are essential plant hormones that influence various developmental processes. Research has shown that root-specific expression of CaCKX6 (chickpea cytokinin oxidase/dehydrogenase) leads to significant increases in lateral root number and root biomass in both Arabidopsis and chickpea plants without negatively affecting vegetative and reproductive shoot growth . This makes CKX6 particularly interesting for agricultural research focused on drought tolerance and root system architecture optimization.

How does CKX6 function at the subcellular level?

Similar to other cytokinin oxidase/dehydrogenase family members like CKX1, CKX6 is likely membrane-associated. Studies with CKX1 have demonstrated localization primarily in the endoplasmic reticulum (ER) . For CaCKX6 specifically, fluorescence overlay analysis and subcellular fractionation experiments have confirmed localization predominantly in the ER, with some minor presence at the plasma membrane . This subcellular distribution is crucial for understanding how CKX6 accesses and degrades cytokinins within plant cells.

What are the common applications for CKX6 antibodies in research?

CKX6 antibodies are valuable tools for studying cytokinin metabolism and signaling in plant biology research. They are commonly used for:

• Protein detection via Western blotting

• Immunohistochemistry to localize CKX6 expression in plant tissues

• Immunoprecipitation for protein interaction studies

• Flow cytometry for quantitative analysis

• Immunofluorescence for subcellular localization studies

What criteria should researchers consider when selecting a CKX6 antibody?

When selecting a CKX6 antibody, researchers should evaluate:

• Antibody specificity: Confirm the antibody recognizes CKX6 without cross-reactivity to other CKX family members

• Host species: Consider compatibility with other antibodies for co-immunostaining experiments

• Clonality: Monoclonal antibodies offer higher specificity but narrower epitope recognition; polyclonal antibodies provide stronger signals but potential batch variability

• Validated applications: Verify the antibody has been successfully used in your intended application (Western blot, IHC, IF, etc.)

• Species reactivity: Ensure the antibody recognizes CKX6 in your experimental organism

Cross-reactivity testing is particularly important, as antibody validation studies for other proteins have shown that unvalidated antibodies can lead to misleading results in research applications .

How can researchers validate the specificity of a CKX6 antibody?

Validation of CKX6 antibody specificity should include:

• Western blot analysis: Confirm a single band of appropriate molecular weight

• Positive and negative controls: Include tissues/cells known to express or lack CKX6

• Knockout/knockdown validation: Test the antibody on samples where CKX6 has been genetically eliminated or reduced

• Epitope competition assay: Pre-incubate the antibody with purified CKX6 protein or peptide to confirm signal disappearance

• Recombinant expression: Test against recombinant CKX6 protein

Similar validation approaches have been used successfully for monoclonal antibodies against other proteins, such as the Nkx6.1 transcription factor, where specificity was confirmed through multiple complementary methods .

What are the optimal conditions for using CKX6 antibodies in Western blotting?

For optimal Western blot results with CKX6 antibodies:

• Sample preparation:

  • For plant tissues, use a buffer containing protease inhibitors to prevent degradation

  • Include reducing agents like DTT or β-mercaptoethanol to break disulfide bonds

  • Heat samples at 95°C for 5 minutes for complete denaturation

• Gel electrophoresis:

  • Use 10-12% polyacrylamide gels for optimal separation

  • Load 20-50 μg of total protein per lane

• Transfer conditions:

  • Transfer to PVDF membrane (preferred over nitrocellulose for plant proteins)

  • Use wet transfer at 100V for 1 hour or 30V overnight at 4°C

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk or BSA in TBST for 1 hour

  • Incubate with primary CKX6 antibody at 1:1000 dilution (optimize as needed)

  • Wash extensively with TBST (3-5 times, 5 minutes each)

  • Use HRP-conjugated secondary antibody at 1:5000-1:10000 dilution

• Detection:

  • Use enhanced chemiluminescence (ECL) for detection

  • For weak signals, consider using signal enhancers or longer exposure times

When interpreting results, be aware that post-translational modifications may cause the observed molecular weight to differ from the calculated value.

How should researchers approach immunolocalization of CKX6 in plant tissues?

For successful immunolocalization of CKX6 in plant tissues:

• Tissue fixation and processing:

  • Fix tissues in 4% paraformaldehyde for 2-4 hours

  • Embed in paraffin or prepare cryosections (10-15 μm thickness)

  • For membrane proteins like CKX6, avoid harsh fixatives that may alter epitope accessibility

• Antigen retrieval:

  • Perform citrate buffer (pH 6.0) heat-induced epitope retrieval

  • Alternative methods include enzymatic retrieval with proteinase K

• Blocking and antibody incubation:

  • Block with 5% normal serum from the species of the secondary antibody

  • Include 0.3% Triton X-100 for membrane permeabilization

  • Incubate with CKX6 primary antibody (1:50-1:200 dilution) overnight at 4°C

  • Wash thoroughly and incubate with fluorophore-conjugated secondary antibody

• Controls:

  • Include tissues known to lack CKX6 expression

  • Perform peptide competition controls

  • Include primary antibody omission controls

Similar immunolocalization approaches have been effectively used for studying subcellular localization of CKX1, another member of the cytokinin oxidase/dehydrogenase family .

How can researchers use co-immunoprecipitation to study CKX6 protein interactions?

Co-immunoprecipitation (Co-IP) is valuable for investigating CKX6 protein-protein interactions:

• Sample preparation:

  • Prepare plant tissue lysates under non-denaturing conditions

  • Use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, and protease inhibitors

• Pre-clearing:

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

  • Incubate for 1 hour at 4°C with gentle rotation

• Immunoprecipitation:

  • Incubate pre-cleared lysate with CKX6 antibody (2-5 μg) overnight at 4°C

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

  • Wash beads 3-5 times with IP buffer

• Analysis:

  • Elute proteins with SDS sample buffer

  • Analyze by Western blot using antibodies against suspected interaction partners

This approach has been successfully used to detect protein-protein interactions of other CKX family members. For example, studies with CKX1 demonstrated homodimerization using co-immunoprecipitation with anti-GFP antibodies in plants co-expressing GFP-CKX1 and myc-CKX1 .

How can researchers utilize CKX6 antibodies in the study of oligomerization?

To study CKX6 oligomerization:

• Blue native PAGE:

  • Solubilize membranes with mild detergents (digitonin or DDM)

  • Separate protein complexes on gradient native gels

  • Transfer to membrane and probe with CKX6 antibody

• Chemical crosslinking:

  • Treat samples with crosslinkers like DSS or formaldehyde

  • Analyze by SDS-PAGE and Western blotting with CKX6 antibody

• Fluorescence resonance energy transfer (FRET):

  • Express CKX6 fused to different fluorophores (e.g., CFP and YFP)

  • Measure FRET signals in planta using confocal microscopy

Research with related proteins has shown that higher order oligomers can be detected via co-immunoprecipitation followed by SDS-PAGE analysis. For instance, studies with CKX1 revealed that myc-CKX1 signals of high molecular mass were most prevalent in the Co-IP fraction, suggesting the formation of stable oligomers that were not fully resolved under SDS-PAGE conditions .

What are common issues when using CKX6 antibodies and how can they be resolved?

How can researchers differentiate between specific and non-specific binding of CKX6 antibodies?

To differentiate between specific and non-specific binding:

• Peptide competition assay: Pre-incubate the antibody with excess purified CKX6 peptide (corresponding to the immunogen) before application to the sample. Specific signals should be eliminated or significantly reduced.

• Knockout/knockdown controls: When available, include samples from CKX6 knockout or knockdown plants as negative controls.

• Isotype controls: For monoclonal antibodies, use an irrelevant antibody of the same isotype and concentration.

• Multiple antibodies approach: Use two different antibodies recognizing distinct epitopes of CKX6. Overlapping signals are more likely to be specific.

• Signal pattern analysis: Specific binding should exhibit consistent localization patterns across technical and biological replicates, matching the expected subcellular distribution (primarily ER localization for CKX6, based on similarities with CKX1) .

How can CKX6 antibodies be used to investigate post-translational modifications?

To investigate post-translational modifications (PTMs) of CKX6:

• Sequential immunoprecipitation:

  • First IP: Use CKX6 antibody to isolate total CKX6 protein

  • Second IP: Use antibodies against specific PTMs (phospho-, glyco-, ubiquitin-specific)

• 2D gel electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Probe with CKX6 antibody to identify different modified forms

• Mass spectrometry analysis:

  • Immunoprecipitate CKX6 using specific antibodies

  • Perform tryptic digestion and analyze by LC-MS/MS

  • Identify PTM sites by analyzing modified peptides

• Phosphorylation-specific analysis:

  • Treat samples with phosphatases before Western blotting

  • Compare migration patterns with and without treatment

This approach has proven valuable for studying post-translational modifications of membrane-localized proteins in plants, particularly those involved in signaling pathways.

How do CKX6 antibodies compare to genetic reporters for studying protein expression?

Comparison between antibody-based detection and genetic reporters:

How might new antibody technologies enhance CKX6 research?

Emerging antibody technologies with potential applications for CKX6 research include:

• Single-domain antibodies (nanobodies):

  • Smaller size allows better penetration into plant tissues

  • Can access epitopes not available to conventional antibodies

  • Potential for in vivo imaging of CKX6 in living plant cells

• Antibody fragments:

  • Fab and scFv fragments offer improved tissue penetration

  • Reduced non-specific binding due to elimination of Fc region

  • Enhanced specificity for challenging applications

• Proximity labeling:

  • Antibody-enzyme fusions (APEX2, BioID) for proximity-dependent labeling

  • Identification of transient CKX6 interaction partners in native context

  • Mapping of protein neighborhoods around CKX6 in the ER membrane

• Super-resolution microscopy compatible antibodies:

  • Direct-labeled antibodies for STORM/PALM imaging

  • Enabling nanoscale visualization of CKX6 distribution in membranes

These technologies could significantly advance our understanding of CKX6 localization and interactions beyond what is possible with conventional antibodies.

What are the methodological considerations for multiplex detection of CKX6 and other proteins?

For multiplex detection of CKX6 alongside other proteins:

• Antibody selection criteria:

  • Choose primary antibodies from different host species

  • Ensure no cross-reactivity between antibodies

  • Validate each antibody individually before multiplexing

• Fluorescent detection optimization:

  • Use fluorophores with minimal spectral overlap

  • Include appropriate controls for autofluorescence

  • Apply spectral unmixing algorithms for closely overlapping signals

• Sequential immunostaining protocol:

  • For same-species antibodies, use sequential immunostaining with intermediate blocking steps

  • Consider tyramide signal amplification for weak signals

  • Employ zenon labeling technology for direct labeling of primary antibodies

• Controls for multiplex staining:

  • Single-antibody controls to verify signal specificity

  • Secondary antibody-only controls to check cross-reactivity

  • Absorption controls with immunizing peptides

Multiplexing approaches allow researchers to study CKX6 in the context of other proteins involved in cytokinin metabolism and signaling pathways, providing more comprehensive insights into functional relationships.