CYCD2-1 Antibody

What is CYCD2-1 and what role does it play in plant cellular processes?

CYCD2-1 (Cyclin D2-1) is a D-type cyclin in Arabidopsis thaliana that plays a crucial role in cell cycle regulation, particularly in promoting the G1-to-S phase transition. It is involved in lateral root initiation through interaction with the inhibitory protein ICK2/KRP2 . When the genomic form of CYCD2-1 is introduced into Arabidopsis, it increases cell formation in the root apex and leaf, raises CDK/CYCLIN enzyme activity, reduces G1-phase duration, and reduces the size of cells at S phase and division . CYCD2-1 functions as part of the regulatory machinery controlling cell proliferation in developing plant tissues, with particular importance in root branching stimulated by auxin .

How does CYCD2-1 localization change throughout the cell cycle?

CYCD2-1 exhibits dynamic localization patterns that are tightly linked to cell cycle progression:

This localization pattern has been confirmed using CYCD2-1-GFP fusion proteins and chromatin markers like H2B-YFP . The nuclear-to-cytoplasmic transition appears to be a critical regulatory mechanism for CYCD2-1 function during cell division cycles.

What experimental systems are optimal for studying CYCD2-1 expression?

For studying CYCD2-1 expression and function, researchers should consider multiple experimental approaches:

• Transgenic Arabidopsis lines expressing CYCD2-1-GFP under its native promoter (ProCYCD2-1:CYCD2-1-GFP), which allows visualization of protein localization in different tissues and cell types

• Root apical and basal meristems, where CYCD2-1 accumulates in all cell files and can be readily observed

• Lateral root initiation sites, where CYCD2-1 interacts with ICK2/KRP2 during auxin-mediated root branching

• Leaf development systems, where CYCD2-1 influences cell proliferation and final cell size

To accurately assess expression, it's essential to use the genomic form of CYCD2-1 rather than cDNA, as the latter may produce aberrantly spliced mRNA that lacks full functionality .

What criteria should guide selection of a CYCD2-1 antibody for plant research?

When selecting a CYCD2-1 antibody for plant research, consider these critical factors:

• Epitope specificity: Antibodies targeting conserved regions of D-type cyclins should be evaluated for cross-reactivity with other cyclins

• Host species: Rabbit-derived monoclonal antibodies often provide high specificity and reproducibility in plant tissue applications

• Validated applications: Confirm the antibody has been validated for your specific application (Western blot, immunohistochemistry, immunofluorescence)

• Species reactivity: Ensure the antibody recognizes plant CYCD2-1 (particularly important when adapting antibodies developed for mammalian systems)

• Clonality: Monoclonal antibodies provide consistent results across experiments and batches

Drawing from approaches used for other cyclins, antibodies targeting the C-terminal region often provide good specificity, as seen with the EPR2241 antibody for Cyclin D1 .

How should researchers validate CYCD2-1 antibody specificity in plant tissues?

Validation should employ multiple complementary approaches:

• Western blot analysis using wild-type and cycd2-1 mutant/knockout tissues to confirm specificity and band size (expected ~34 kDa by comparison with other D-type cyclins)

• Immunoprecipitation followed by mass spectrometry to confirm the antibody captures authentic CYCD2-1

• Immunofluorescence co-localization with CYCD2-1-GFP fusion proteins

• Competitive binding assays with purified CYCD2-1 protein

• Biophysical quality control to confirm antibody identity at molecular level

• Analysis of CYCD2-1 levels in tissues where expression is known to change (e.g., during lateral root development)

These validation steps are essential as non-specific binding can lead to misinterpretation of experimental results, particularly in developmental studies where cyclins show dynamic expression patterns.

What are the most effective positive and negative controls for CYCD2-1 antibody experiments?

Robust experimental design requires appropriate controls:

Positive controls:

• Tissues with known high CYCD2-1 expression (root apical meristem, young leaf primordia)

• Recombinant CYCD2-1 protein

• Transgenic plants overexpressing CYCD2-1

Negative controls:

• cycd2-1 knockout/knockdown plant tissues

• Secondary antibody-only controls to assess background staining

• Tissues with minimal CYCD2-1 expression (mature elongated cells)

• Pre-immune serum controls

• Peptide competition assays where the antibody is pre-incubated with the immunizing peptide

Using tissues at different developmental stages can provide internal controls within the same experiment, as CYCD2-1 expression varies with cell differentiation status .

What are the optimal methods for detecting CYCD2-1 in plant tissues by Western blot?

For successful Western blot detection of CYCD2-1 in plant tissues:

• Sample preparation:

  • Harvest young, actively dividing tissues (meristems, young leaves)

  • Extract proteins using RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors

  • Include 10-20 μg of total protein per lane

• Electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels under reducing conditions

  • Transfer to PVDF membranes at 100V for 60-90 minutes in cold transfer buffer

• Antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with primary CYCD2-1 antibody (optimal dilution must be determined empirically, typically 1:1000-1:10000)

  • Wash thoroughly with TBST (3-5 washes, 5 minutes each)

  • Incubate with HRP-conjugated secondary antibody at 1:1000-1:5000 dilution

• Detection:

  • Visualize using ECL development solution

  • Expected band size should be approximately 34 kDa (by comparison with other D-type cyclins)

When analyzing CYCD2-1 protein levels, include loading controls such as alpha-tubulin or actin, and consider using phospho-specific antibodies if investigating CYCD2-1 phosphorylation status.

How can immunolocalization techniques be optimized for CYCD2-1 detection in fixed plant tissues?

For optimal immunolocalization of CYCD2-1:

• Tissue fixation options:

  • 4% paraformaldehyde in PBS (pH 7.4) for 2-4 hours at room temperature

  • 100% methanol for 5 minutes at room temperature for cultured cells

  • Alternatively, use an ethanol:acetic acid (3:1) mixture for plant tissues

• Antigen retrieval:

  • Heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes

  • Allow sections to cool slowly to room temperature

• Blocking and antibody incubation:

  • Block with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour

  • Incubate with primary antibody overnight at 4°C

  • Perform multiple washes with PBS-T

  • Incubate with fluorophore-conjugated secondary antibody for 1-2 hours at room temperature

• Counterstaining:

  • Use DAPI for nuclear staining

  • Consider co-staining with cell wall markers (calcofluor white) or other cellular markers

• Controls:

  • Include peptide-competed antibody control

  • Secondary antibody-only control

  • Comparison with CYCD2-1-GFP localization patterns

This approach should reveal the dynamic nuclear-to-cytoplasmic transitions of CYCD2-1 during cell cycle progression .

What cell synchronization methods are most effective when studying CYCD2-1 dynamics?

To study CYCD2-1 dynamics throughout the cell cycle:

• Hydroxyurea synchronization:

  • Treat cell cultures or root tips with 5-10 mM hydroxyurea for 12-24 hours to arrest cells at G1/S boundary

  • Release by washing and transferring to fresh media

  • Collect samples at regular intervals (30 min to 2 hours) to capture different cell cycle phases

• Aphidicolin treatment:

  • Inhibits DNA polymerase α, arresting cells at G1/S transition

  • Use 5-10 μg/ml for 12-24 hours, then release

• Oryzalin or propyzamide treatment:

  • Microtubule-disrupting agents that arrest cells in M-phase

  • Remove drug and collect samples during recovery to observe re-establishment of CYCD2-1 nuclear localization

• BrdU pulse labeling:

  • Label S-phase cells to correlate with CYCD2-1 localization

  • Allows measurement of cell size at S-phase, which is reduced (28% smaller) in CYCD2-1 overexpressing plants

• Flow cytometry validation:

  • Confirm synchronization by analyzing the G1/G2 ratio

  • CYCD2-1 overexpression reduces this ratio from 1.31 to 0.78, indicating accelerated exit from G1 phase

These approaches help correlate CYCD2-1 localization with specific cell cycle phases, revealing its role in regulating the G1/S transition.

How can researchers investigate CYCD2-1 and ICK2/KRP2 protein interactions?

To study the regulatory interaction between CYCD2-1 and its inhibitor ICK2/KRP2:

• Co-immunoprecipitation:

  • Immunoprecipitate with anti-CYCD2-1 antibody

  • Blot for ICK2/KRP2, or vice versa

  • Include controls with non-specific IgG

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse CYCD2-1 and ICK2/KRP2 to complementary fragments of a fluorescent protein

  • Co-expression will generate fluorescence signal when proteins interact

  • This allows visualization of interaction sites within plant cells

• Yeast two-hybrid assays:

  • Confirm direct protein-protein interaction

  • Map interaction domains through deletion constructs

• FRET-FLIM analysis:

  • Use fluorescently tagged proteins to measure energy transfer

  • Provides quantitative measurement of protein proximity in living cells

• Proximity ligation assay:

  • Allows visualization of endogenous protein interactions in situ

  • Particularly useful for rare or transient interactions

Since auxin reduces ICK2/KRP2 protein levels, which affects both activity and cellular distribution of CYCD2-1 , these methods can reveal how environmental signals modulate this regulatory interaction.

What approaches can be used to analyze CYCD2-1 phosphorylation status and its functional significance?

To investigate CYCD2-1 phosphorylation:

• Phospho-specific antibodies:

  • Develop antibodies against predicted phosphorylation sites

  • Use bioinformatics to identify conserved CDK phosphorylation motifs

• Phosphatase treatment experiments:

  • Treat protein extracts with lambda phosphatase before Western blot

  • Compare mobility shifts with untreated samples

• Phos-tag SDS-PAGE:

  • Enhances separation of phosphorylated from non-phosphorylated proteins

  • Allows visualization of multiple phosphorylation states

• Mass spectrometry analysis:

  • Perform immunoprecipitation of CYCD2-1

  • Analyze by LC-MS/MS to identify phosphorylation sites

  • Compare samples from different cell cycle phases or treatments

• Phospho-mimetic and phospho-null mutations:

  • Generate S/T→E/D (mimics phosphorylation) or S/T→A (prevents phosphorylation) variants

  • Express in cycd2-1 mutant background to assess functional significance

  • Analyze effects on protein localization, stability, and cell cycle progression

These approaches will help determine how phosphorylation regulates CYCD2-1 activity and its interaction with ICK2/KRP2 during cell cycle progression.

How can multi-omics approaches be integrated to understand CYCD2-1 regulatory networks?

Integrating multiple omics approaches:

• Transcriptomics:

  • RNA-seq of wild-type vs. cycd2-1 mutants or CYCD2-1 overexpressors

  • Identify genes co-regulated with CYCD2-1 during development

  • Compare expression profiles across tissues and developmental stages

• Proteomics:

  • Immunoprecipitation coupled with mass spectrometry to identify CYCD2-1 interactors

  • Quantitative proteomics to measure protein abundance changes

  • Phosphoproteomics to map signaling networks

• Chromatin immunoprecipitation (ChIP-seq):

  • Identify genomic regions associated with CYCD2-1-containing complexes

  • Map E2F transcription factor binding sites regulated by CYCD2-1/CDK activity

• Metabolomics:

  • Profile metabolic changes in CYCD2-1 mutants

  • Investigate links between carbon metabolism and cell cycle control

• Computational integration:

  • Network analysis to identify regulatory hubs

  • Pathway enrichment to understand biological processes affected

  • Machine learning approaches to predict new regulatory connections

This integrated approach can reveal how CYCD2-1 functions within the broader context of plant development, particularly in auxin-mediated lateral root initiation where ICK2/KRP2 restrains root ramification by maintaining CYCD2-1 inactive .

How should researchers interpret varied CYCD2-1 localization patterns across different cell types?

When encountering variable CYCD2-1 localization patterns:

• Cell cycle phase considerations:

  • CYCD2-1 shows nuclear localization in G1 but becomes cytoplasmic before mitosis

  • Asynchronous cell populations will naturally show mixed localization patterns

  • Correlate with cell cycle markers (e.g., H2B-YFP intensity for DNA content)

• Tissue-specific regulation:

  • In most meristem tissues, CYCD2-1 is predominantly nuclear

  • Signal becomes weaker in elongating cortical and epidermal cells

  • Quiescent center and adjacent initials show weaker/more diffuse signal

• Developmental context:

  • CYCD2-1 localization changes during cell differentiation

  • Different cell types may have distinct CYCD2-1 regulatory mechanisms

• Auxin effects:

  • Auxin reduces ICK2/KRP2 protein levels, affecting both activity and localization of CYCD2-1

  • Auxin gradients in tissues may explain localization differences

• Technical considerations:

  • Fixation artifacts can alter apparent protein localization

  • GFP fusion proteins may have subtly different localization than endogenous proteins

  • Antibody accessibility issues in different tissues

Remember that dynamic localization is likely a key regulatory mechanism for CYCD2-1 function, with nuclear import/export serving as control points during cell cycle progression.

What approaches help resolve inconsistent Western blot results when detecting CYCD2-1?

To resolve inconsistent Western blot results:

• Protein extraction optimization:

  • Use multiple extraction buffers (RIPA, urea-based, phenol-based) to compare efficiency

  • Include protease and phosphatase inhibitors to prevent degradation

  • Extract from tissues with known high CYCD2-1 expression (meristems, young leaves)

• Sample handling:

  • Maintain samples at 4°C throughout preparation

  • Add sample buffer immediately after extraction and heat promptly

  • Avoid repeated freeze-thaw cycles

• Blocking optimization:

  • Test different blocking agents (BSA vs. non-fat dry milk)

  • Optimize blocking time and temperature

  • Consider alternative blocking buffers for phospho-specific detection

• Antibody validation:

  • Test antibody specificity with recombinant CYCD2-1

  • Perform peptide competition assays

  • Use multiple antibodies targeting different epitopes if available

• Detection system troubleshooting:

  • Compare chemiluminescent, fluorescent, and colorimetric detection methods

  • Optimize exposure times to avoid saturation

  • Include positive control samples (e.g., CYCD2-1 overexpressing tissues)

Remember that CYCD2-1 may be present as full-length protein and shorter forms, as observed with CYCD2-1-GFP fusion proteins that showed both the expected 75 kDa band and shorter proteins recognized by anti-CYCD2-1 antiserum .

How can researchers differentiate between splice variants and post-translational modifications of CYCD2-1?

To distinguish between splice variants and post-translational modifications:

• Transcript analysis:

  • Perform RT-PCR with primers spanning expected splice junctions

  • Sequence cDNA to identify alternative splice forms

  • Compare with genomic sequence to confirm intron retention or alternative splicing

  • Note that cDNA forms of CYCD2-1 can produce aberrantly spliced mRNAs compared to genomic forms

• Protein analysis:

  • Use epitope-specific antibodies targeting different regions of CYCD2-1

  • Compare migration patterns on standard vs. Phos-tag gels

  • Treat samples with phosphatase, glycosidase, or other enzymes before Western blot

• Mass spectrometry approaches:

  • Perform top-down proteomics to identify intact protein forms

  • Use bottom-up proteomics with high sequence coverage to map modifications

  • Compare peptide maps to predicted splice variant sequences

• Functional studies:

  • Express different splice variants as tagged proteins

  • Compare their localization, interaction partners, and activity

  • Assess impact on cell cycle progression and plant development

When analyzing Western blots, note that researchers observed both full-length CYCD2-1-GFP (75 kDa) and shorter proteins recognized by anti-CYCD2-1 antiserum that were not detected by anti-GFP antibodies , suggesting possible processing or alternative forms of the protein.