CYCB2-2 Antibody

What is CYCB2-2 and what is its role in cell cycle regulation?

CYCB2-2 belongs to the B-type cyclin family, which are essential components of the cell cycle regulatory machinery. B-type cyclins associate with p34cdc2 (a cyclin-dependent kinase) to form complexes that regulate mitotic progression. Specifically, CYCB2-2 is involved in both mitotic cell cycle and endoreduplication phases in plant development, particularly in maize endosperm development . In mammalian systems, Cyclin B2 plays a key role in transforming growth factor beta-mediated cell cycle control through its binding to transforming growth factor beta RII . Unlike its counterpart Cyclin B1 that co-localizes with microtubules, Cyclin B2 is primarily associated with the Golgi region, suggesting distinctive roles for these two B-type cyclins .

How does CYCB2-2 localization change during different cell cycle phases?

CYCB2-2 demonstrates dynamic localization patterns that correlate with specific cell cycle phases. During mitotic division in plants, CYCB2-2 is generally localized to the nucleus of endosperm cells, but interestingly, a lower molecular weight form of the protein accumulates specifically in the cytosol of endoreduplicating endosperm cells . In dividing cells, CYCB2-2 has been observed to localize to the phragmoplast, suggesting involvement in cytokinesis and cell wall formation processes . This localization pattern differs from mammalian Cyclin B2, which shows Golgi-region association . These distinct localization patterns provide important insights into the functional specialization of this cyclin during different developmental processes.

What is the molecular weight range for detecting CYCB2-2 in various species?

The expected molecular weight for detecting CYCB2-2 varies slightly depending on the species and possible post-translational modifications. In mammalian systems, Cyclin B2 appears as a prominent band at approximately 51 kDa in Western blot analyses . Some antibodies may detect additional bands, such as the ~80 kDa band observed with MA1-156 antibody . The calculated molecular weight for human CCNB2 is 45282 Da according to antibody documentation . In maize, CYCB2-2 detection shows the expected molecular weight band, but longer autoradiograph exposure times reveal an additional band of slightly lower molecular weight that accumulates specifically during the endoreduplication phase of endosperm development (detectable from around 13-15 DAP) .

What criteria should guide the selection of an appropriate anti-CYCB2-2 antibody for my research?

When selecting an anti-CYCB2-2 antibody, consider these critical factors:

• Species reactivity: Verify that the antibody reacts with your study organism. For example, MA1-156 detects Cyclin B2 in Xenopus laevis and mammalian sources , while A02040-2 reacts with human, mouse, and rat CCNB2 .

• Application compatibility: Confirm the antibody is validated for your intended applications. For instance, MA1-156 has been successfully used in immunofluorescence, immunoprecipitation, IHC(P), and Western blot procedures .

• Epitope specificity: Consider the epitope targeted by the antibody to avoid cross-reactivity. For maize CYCB2-2 studies, antibodies were raised against the N-terminal region (amino acid residues 4-143) that has little sequence identity with other maize cyclins (~39% identity with CYCB2;1) .

• Clonality: Monoclonal antibodies like MA1-156 offer consistent results across experiments, while polyclonal antibodies like A02040-2 potentially recognize multiple epitopes.

• Validated controls: Review the literature or manufacturer data showing antibody specificity against known positive and negative controls.

How can I validate the specificity of a CYCB2-2 antibody?

Thorough validation ensures reliable experimental results. Implement these validation approaches:

• Western blot analysis: Confirm the antibody detects a band of expected molecular weight (~51 kDa for mammalian Cyclin B2) . Test across multiple cell types or tissues with known differential expression of CYCB2-2.

• Recombinant protein controls: Test antibody reactivity against recombinant CYCB2-2 and related cyclins. For example, maize CYCB2-2 antibodies were tested against recombinant CYCB2-2 antigen and comparable N-terminal regions of maize CYCA1;1 and CYCB1;3 expressed as GST-fusions .

• Immunodepletion: Pre-absorb the antibody with its specific antigen to confirm staining specificity.

• Knockout/knockdown controls: When available, use CYCB2-2 knockout/knockdown samples as negative controls.

• Subcellular localization verification: Confirm that immunostaining patterns match the expected localization (e.g., Golgi association for mammalian Cyclin B2 or nuclear/phragmoplast localization for plant CYCB2-2 ).

What controls are essential when using CYCB2-2 antibodies in immunolocalization experiments?

For robust immunolocalization experiments with CYCB2-2 antibodies, include these essential controls:

• Co-staining with subcellular markers: Use markers for relevant structures (DNA stain for nuclei, tubulin for microtubules/phragmoplast) to confirm the expected spatial relationship of CYCB2-2 with cellular compartments.

• Cell cycle phase controls: Include samples from different cell cycle phases to verify phase-specific localization patterns.

• Primary antibody omission: To assess background from secondary antibody.

• Secondary antibody specificity control: Use secondary antibody only on samples without primary antibody treatment.

• Pre-immune serum control: For polyclonal antibodies, use pre-immune serum at equivalent concentration to antibody.

• Tissue-specific expression control: Include tissues known to express high and low levels of CYCB2-2, based on expression data like that compiled from Nimblegen-derived RNA expression data .

What are the optimal conditions for Western blotting with CYCB2-2 antibodies?

For optimal Western blot detection of CYCB2-2:

• Sample preparation: Extract proteins under conditions that preserve phosphorylation states, as cyclin activity is regulated by phosphorylation. For plant samples, follow extraction protocols as described by Sabelli et al. (2013) and Dante et al. (2014) .

• Antibody dilution:

  • For polyclonal antibodies like A02040-2, use at 1:1,000 dilution

  • For monoclonal antibodies, follow manufacturer's recommendations

• Detection of isoforms: Use gradient gels (7-15%) to resolve potential isoforms, particularly when studying developmental transitions like endoreduplication where lower molecular weight forms may appear .

• Controls: Include recombinant CYCB2-2 protein as a positive control and extracts from tissues with low CYCB2-2 expression as negative controls.

• Multiple exposure times: Use various exposure times to detect less abundant isoforms, as demonstrated in studies where longer autoradiograph exposure times revealed an additional band of slightly lower molecular weight during endoreduplication .

How should I optimize immunoprecipitation protocols for studying CYCB2-2 protein interactions?

To successfully immunoprecipitate CYCB2-2 and its interacting partners:

• Antibody selection: Choose antibodies that don't interfere with protein-protein interactions. For CYCB2-2, antibodies raised against the N-terminal region lying outside the Cyc_N domain won't likely interfere with catalytic or CDK-binding activity .

• Buffer optimization: Use buffers that maintain native protein conformation and interactions. Follow established protocols like those described by Dante et al. (2014) .

• Pre-clearing samples: Remove proteins that bind non-specifically to beads by pre-clearing lysates with beads alone.

• Validation of interactions: Confirm interactions by reciprocal co-immunoprecipitation using antibodies against suspected binding partners (e.g., anti-PSTAIR antibody for maize CDKs) .

• Functional validation: Test immunoprecipitates for kinase activity on substrates like histone H1 in in vitro assays, as performed for CYCB2-2 .

• Controls: Include isotype-matched control antibodies to identify non-specific binding.

What approaches work best for studying CYCB2-2 kinase activity?

To effectively study CYCB2-2-associated kinase activity:

• Immunoprecipitation-kinase assay: Immunoprecipitate CYCB2-2 from cell lysates and assay the precipitate for kinase activity on substrates like histone H1, following protocols described by Dante et al. (2014) .

• Developmental timing considerations: Compare kinase activity across developmental stages. For example, kinase activity associated with CYCB2-2 in mitotic endosperm was found to be absent or greatly reduced in immature ear and endoreduplicating endosperm .

• Co-expression systems: Express CYCB2-2 with potential CDK partners in heterologous systems (like Drosophila S2 cells) to study specific interactions and resulting kinase activity .

• Inhibitor studies: Use specific CDK inhibitors to confirm the type of kinase activity associated with immunoprecipitated CYCB2-2.

• Substrate specificity: Test multiple substrates to characterize the specificity of CYCB2-2-associated kinase activity.

How should I interpret multiple bands in Western blots when using CYCB2-2 antibodies?

Multiple bands in CYCB2-2 Western blots require careful interpretation:

• Expected isoforms: A lower molecular weight CYCB2-2-related polypeptide appears specifically during the endoreduplication phase of maize endosperm development (from around 13-15 DAP), suggesting a functional significance in this specialized cell cycle .

• Cross-reactivity consideration: Some antibodies like MA1-156 cross-react with Cyclin B1 in Western blot applications , so confirm band identity with additional techniques.

• Degradation products: Cyclins undergo regulated degradation during cell cycle progression; multiple bands may represent intermediates in this process. Proper sample handling and protease inhibitors help distinguish true isoforms from degradation artifacts.

• Post-translational modifications: Phosphorylation and other modifications can create band shifts. Compare with phosphatase-treated samples to identify modification-dependent mobility shifts.

• Unexpected bands: MA1-156 detects an unknown band at ~80 kDa in addition to the expected ~51 kDa band . Document these consistently appearing bands and investigate their identity through additional techniques.

What are common challenges in immunolocalization of CYCB2-2 and how can I address them?

Researchers frequently encounter these challenges when immunolocalizing CYCB2-2:

• Low signal intensity:

  • Solution: Optimize antibody concentration (typically 0.5-1 μg/ml for immunohistochemical localization)

  • Enhance signal using amplification systems

  • Increase incubation time at 4°C

• Background fluorescence:

  • Solution: Thoroughly block with appropriate blocking buffer

  • Include detergents in washing steps

  • Use highly-purified antibody preparations like affinity-purified antibodies

• Cell cycle-dependent expression:

  • Solution: Synchronize cells or use markers to identify cell cycle phases

  • Compare with RNA expression data across developmental stages

• Fixation artifacts:

  • Solution: Compare multiple fixation protocols for optimal epitope preservation

  • Follow established protocols successful for CYCB2-2 localization

• Co-localization challenges:

  • Solution: Use high-resolution confocal microscopy

  • Apply appropriate controls for spectral bleed-through

What might cause contradictory results between RNA expression and protein detection of CYCB2-2?

Discrepancies between CYCB2-2 RNA and protein levels can occur for several reasons:

• Post-transcriptional regulation: In maize endosperm, CYCB2-2 RNA levels decline as the endosperm transitions from mitotic to endoreduplication cell cycle, yet protein levels remain relatively constant . This suggests post-transcriptional regulation mechanisms.

• Protein stability differences: CYCB2-2 was found to be recalcitrant to degradation by the 26S proteasome in endoreduplicating endosperm extracts, potentially explaining its sustained accumulation despite declining RNA levels .

• Cell cycle-specific regulation: Cyclins typically show cell cycle-dependent expression; asynchronous cell populations may show mismatches between RNA sampling and protein detection.

• Methodological considerations:

  • Confirm RNA analysis with multiple techniques (RT-PCR, RNA-seq)

  • Validate protein detection with different antibodies

  • Use absolute quantification methods when possible

• Tissue heterogeneity: In developing organs like maize endosperm, different cell populations may be in different states, affecting bulk measurements of RNA vs. protein.

How can CYCB2-2 antibodies be used to study specialized cell cycles like endoreduplication?

CYCB2-2 antibodies offer valuable insights into specialized cell cycles through these approaches:

• Isoform tracking: Monitor the appearance of the lower molecular weight CYCB2-2-related polypeptide that accumulates specifically during endoreduplication in maize endosperm .

• Subcellular localization changes: Track CYCB2-2 relocalization from nucleus to cytoplasm during the transition to endoreduplication, as the lower molecular weight form accumulates specifically in the cytosol of endoreduplicating endosperm cells .

• Kinase activity correlation: Compare CYCB2-2-associated kinase activity between mitotic tissues and endoreduplicating tissues to identify regulatory differences. This is particularly important as kinase activity was found to be associated with CYCB2-2 in mitotic endosperm but absent or greatly reduced in endoreduplicating endosperm .

• Quantitative immunofluorescence: Measure CYCB2-2 levels in individual nuclei and correlate with DNA content to assess relationships with endoreduplication cycles.

• Proteasome sensitivity assays: Compare degradation patterns of CYCB2-2 between mitotic and endoreduplicating extracts to understand differential regulation, building on findings that CYCB2-2 was recalcitrant to degradation by the 26S proteasome in endoreduplicating endosperm extracts .

What approaches can be used to investigate CYCB2-2 interactions with cell division structures?

To explore CYCB2-2 interactions with cellular structures:

• Co-immunolocalization with structural markers: Combine CYCB2-2 antibodies with markers for cellular structures like phragmoplast and cell plate formation components, expanding on observations that CYCB2-2 appears to be localized to the phragmoplast in dividing cells .

• Live-cell imaging: Develop fluorescently-tagged CYCB2-2 constructs to visualize dynamic interactions during cell division.

• Proximity ligation assays: Detect in situ interactions between CYCB2-2 and suspected binding partners with spatial resolution.

• Immunoelectron microscopy: Precisely localize CYCB2-2 relative to subcellular structures with nanometer resolution.

• BiFC or FRET analyses: Investigate direct protein-protein interactions between CYCB2-2 and structural components of the cell division machinery.

How can CYCB2-2 antibodies contribute to understanding evolutionary conservation of cell cycle regulation?

CYCB2-2 antibodies can reveal evolutionary insights through:

• Cross-species application: Test antibody reactivity across diverse species to identify conserved epitopes. For example, some antibodies show reactivity across mammalian species and Xenopus .

• Comparative localization studies: Compare subcellular localization patterns between plant and animal systems. This is particularly interesting given that plant CYCB2-2 shows nuclear and phragmoplast association , while mammalian Cyclin B2 associates with the Golgi region .

• Functional domain mapping: Use domain-specific antibodies to identify conserved functional domains across species. For example, antibodies raised against the N-terminal region of maize CYCB2-2 lying outside the Cyc_N domain could be used to explore equivalent regions in other species.

• Interaction network comparison: Analyze CYCB2-2 binding partners across species through immunoprecipitation followed by mass spectrometry.

• Differential regulation analysis: Compare cell cycle-dependent degradation patterns of CYCB2-2 across evolutionarily diverse systems to identify conserved regulatory mechanisms.