CDKC-2 Antibody

What is CDK2 and what cellular functions does it regulate?

CDK2 is a 33-34 kDa enzyme encoded by the CDK2 gene located on Chromosome 12 in humans. It functions as a catalytic subunit of the cyclin-dependent kinase complex, with activity primarily restricted to the G1-S phase of the cell cycle. CDK2 is essential for the cellular G1/S transition, playing a critical role in controlling cell proliferation . Studies indicate that overexpression of CDK2 may cause abnormal regulation of the cell cycle, potentially contributing to the hyperproliferation of cancerous cells. Additionally, CDK2 regulates HOX genes, which play key roles in cell differentiation and morphogenesis .

How do researchers distinguish between CDK2 and related cyclin-dependent kinases in experimental settings?

Distinguishing CDK2 from other CDKs requires careful antibody selection and validation. Western blot analysis using specific CDK2 antibodies can detect a band at approximately 34 kDa under reducing conditions, while related proteins such as CDK4 and CDK6 appear at different molecular weights . When using CDK2 antibodies, it's advisable to include positive controls with recombinant CDK2, CDK4, and CDK6 to confirm specificity. The AF4654 antibody, for example, has demonstrated specific detection of CDK2 without cross-reactivity to other CDK family members in Western blot applications .

What is the difference between CDKC;2 in plants and CDK2 in mammals?

While both are cyclin-dependent kinases, CDKC;2 and CDK2 serve distinct functions in their respective organisms. CDKC;2 in plants like Arabidopsis phosphorylates the RNA polymerase II C-terminal domain (Pol II CTD) and influences circadian clock periods . In contrast, mammalian CDK2 primarily regulates the G1/S phase transition in the cell cycle. Structurally, plant CDKC;2 shares more homology with human CDK9 than with CDK2, as evidenced by molecular modeling studies using the human CDK9-cyclinT1 complex as a template for predicting the structure of the CDKC;2-CYCT1;4 complex .

What are the optimal protocols for using CDK2 antibodies in Western blot applications?

For optimal Western blot detection of CDK2, researchers should consider the following methodology:

• Sample preparation: Prepare cell or tissue lysates under reducing conditions using appropriate lysis buffers (e.g., Immunoblot Buffer Group 1)

• Protein loading: 0.2 mg/mL of lysate is typically sufficient for detection

• Membrane type: PVDF membranes provide good results for CDK2 detection

• Antibody concentration: Use 1 μg/mL of CDK2-specific antibody (e.g., Goat Anti-Human/Mouse CDK2 Antigen Affinity-purified Polyclonal Antibody)

• Secondary antibody: HRP-conjugated species-appropriate secondary antibodies (e.g., HRP-conjugated Anti-Goat IgG)

• Expected band size: Approximately 34-38 kDa, depending on the cell type and detection system

The specific protocol may need optimization depending on the particular antibody used and the experimental system.

What immunohistochemistry methods provide optimal CDK2 detection in tissue samples?

For effective immunohistochemical detection of CDK2 in tissue samples:

• Fixation: Use immersion-fixed paraffin-embedded sections

• Antigen retrieval: Optimize based on tissue type (typically heat-mediated citrate buffer)

• Antibody concentration: 3-5 μg/mL for primary CDK2 antibody is effective for most tissue types

• Incubation time: 1 hour at room temperature works well for many applications

• Detection system: HRP polymer antibody systems (e.g., Anti-Goat IgG VisUCyte HRP Polymer Antibody) provide sensitive detection

• Visualization: DAB (brown) for chromogenic detection with hematoxylin (blue) counterstain

For lung cancer tissue, CDK2 staining is typically localized to the cytoplasm and cancer cell nuclei . For other tissue types, protocol optimization may be necessary.

How can researchers optimize immunofluorescence protocols for CDK2 detection in different cell types?

Immunofluorescence detection of CDK2 requires different approaches depending on whether cells are adherent or in suspension:

For adherent cells (e.g., 3T3-L1 mouse cells):

• Fixation: Immersion fixation of cells grown on coverslips

• Antibody concentration: 15 μg/mL of CDK2 antibody

• Incubation: 3 hours at room temperature

• Detection: Fluorochrome-conjugated secondary antibody (e.g., NorthernLights 557-conjugated Anti-Goat IgG)

• Counterstain: DAPI for nuclear visualization

• Expected pattern: Cytoplasmic and nuclear localization

For non-adherent cells (e.g., K562 cells):

• Fixation: Immersion fixation of cells in suspension

• Antibody concentration: 5 μg/mL of CDK2 antibody

• Incubation: 3 hours at room temperature

• Detection: Same fluorescent secondary antibody system

• Expected pattern: Primarily cytoplasmic localization

How can CDK2 antibodies be utilized to investigate cell cycle dysregulation in cancer research?

CDK2 antibodies serve as valuable tools for investigating cell cycle dysregulation in cancer through several advanced applications:

• Comparative expression analysis: CDK2 antibodies can detect differential expression between normal and cancerous tissues. In human lung cancer tissue, for example, CDK2 antibodies reveal specific staining in cancer cell nuclei, indicating altered localization compared to normal tissues .

• Cell cycle checkpoint analysis: By combining CDK2 antibodies with markers of other cell cycle regulators (cyclins, CDK inhibitors), researchers can elucidate mechanisms of G1/S checkpoint dysregulation in cancer cells.

• Therapeutic response assessment: CDK2 antibodies can monitor changes in CDK2 expression/activity following treatment with cell cycle inhibitors. For instance, in HPV-negative head and neck squamous cell carcinoma, cisplatin exposure causes c-Myc-dependent resistance to CDK4/6 inhibition, which can be analyzed using CDK2 antibodies to understand compensatory mechanisms .

• HOXA7-CDK2 pathway investigation: Studies have demonstrated that HOXA7-promoted cell proliferation (mediated by cyclin E1/CDK2) occurs in hepatocellular carcinoma, making CDK2 antibodies valuable markers for investigating this oncogenic pathway .

What approaches can researchers use to study post-translational modifications of CDK2?

Studying post-translational modifications (PTMs) of CDK2 requires specialized techniques utilizing antibodies:

• Phospho-specific antibodies: Use antibodies that recognize specific phosphorylated residues of CDK2 (e.g., Thr160) to assess activation status.

• Sequential immunoprecipitation:

  • First immunoprecipitate CDK2 using a total CDK2 antibody

  • Then probe with antibodies against specific modifications (phosphorylation, ubiquitination, SUMOylation)

  • Alternatively, first immunoprecipitate with PTM-specific antibodies, then probe with CDK2 antibodies

• 2D gel electrophoresis: Combine with Western blotting using CDK2 antibodies to separate differentially modified forms of CDK2 based on isoelectric point shifts caused by PTMs.

• Mass spectrometry validation: Use CDK2 antibodies for immunoprecipitation followed by mass spectrometry to comprehensively identify all modifications on the protein.

• Proximity ligation assays: Combine CDK2 antibodies with antibodies against specific modifying enzymes to visualize interactions and modification events in situ.

How do expression patterns of CDK2 differ between cell types and how can this be quantified?

CDK2 expression and localization patterns vary significantly across cell types and can be quantified using antibody-based approaches:

Quantification methods include:

• Western blot densitometry normalized to housekeeping proteins

• Immunofluorescence intensity measurements using digital image analysis

• Flow cytometry with permeabilized cells for intracellular CDK2 quantification

• High-content imaging systems for automated quantification of nuclear/cytoplasmic ratios

What are common causes of non-specific binding with CDK2 antibodies and how can they be mitigated?

Non-specific binding is a common challenge when working with CDK2 antibodies. Researchers can address this issue through several strategies:

• Antibody validation: Verify antibody specificity using positive controls (recombinant CDK2) and negative controls (CDK2 knockout cells or tissues) .

• Blocking optimization:

  • Increase blocking time or concentration (typically 5% BSA or 5% non-fat dry milk)

  • Consider species-specific blocking reagents when appropriate

  • Use commercial blocking solutions specifically designed to reduce background

• Antibody dilution: Determine optimal dilutions for each application through titration experiments. For Western blots, 1 μg/mL is often effective for CDK2 detection .

• Secondary antibody selection: Choose highly cross-adsorbed secondary antibodies to minimize species cross-reactivity.

• Sample preparation: Ensure complete lysis and denaturation for Western blot applications, and appropriate fixation for immunohistochemistry or immunofluorescence.

• Preabsorption controls: Preincubate CDK2 antibody with recombinant CDK2 protein before application to verify that staining is eliminated, confirming specificity.

How can researchers address inconsistent results when detecting CDK2 across different experimental systems?

Inconsistent results with CDK2 antibodies across different experimental systems can be addressed through systematic troubleshooting:

• Protocol standardization:

  • Maintain consistent sample preparation methods

  • Standardize antibody concentrations (1 μg/mL for Western blot, 3-15 μg/mL for IHC/IF)

  • Use consistent incubation times and temperatures

• Antibody storage and handling:

  • Store antibodies according to manufacturer recommendations (-20 to -70°C for long-term storage)

  • Avoid repeated freeze-thaw cycles (limit to 1 month at 2-8°C under sterile conditions after reconstitution)

  • Centrifuge concentrated antibodies prior to use

• Species considerations:

  • Verify cross-reactivity with your species of interest (human, mouse, rat)

  • Use species-appropriate positive controls

• Cell cycle synchronization:

  • CDK2 expression and localization vary throughout the cell cycle

  • Synchronize cells when comparing treatments or conditions

  • Document cell confluence and culture conditions

• Quantification standardization:

  • Use consistent exposure times for imaging

  • Include calibration standards for quantitative Western blots

  • Apply consistent analysis parameters in image quantification software

What considerations are important when selecting a CDK2 antibody for multi-species research?

When conducting research across multiple species, antibody selection becomes critically important:

• Epitope conservation analysis:

  • Verify sequence homology of the immunogen across target species

  • CDK2 is highly conserved between human, mouse, and rat, making many antibodies suitable for cross-species work

• Validated cross-reactivity:

  • Select antibodies explicitly validated in multiple species (e.g., AF4654 is validated for human, mouse, and rat CDK2)

  • Review published literature for successful use in your species of interest

• Application-specific validation:

  • An antibody that works for Western blot in multiple species may not work equally well for IHC across species

  • Perform separate validations for each application in each species

• Control samples:

  • Include species-specific positive controls (e.g., HEK293 for human, Balb/3T3 for mouse, C6 for rat)

  • Consider recombinant proteins as standards when comparing across species

• Species-specific secondary antibodies:

  • Use secondary antibodies appropriate for each species' primary antibody

  • Ensure secondary antibodies do not cross-react with endogenous immunoglobulins in your samples

How can CDK2 antibodies be utilized in studying drug resistance mechanisms in cancer?

CDK2 antibodies are valuable tools for investigating drug resistance mechanisms in cancer therapy:

• CDK inhibitor resistance studies: Research demonstrates that cisplatin exposure causes c-Myc-dependent resistance to CDK4/6 inhibition in HPV-negative head and neck squamous cell carcinoma, a mechanism that can be elucidated using CDK2 antibodies to track compensatory activation of alternative CDK pathways .

• Cell cycle checkpoint adaptation: CDK2 antibodies can detect alterations in checkpoint control after chemotherapy treatment. For instance, differential cell cycle arrest in B cell lymphomas affects sensitivity to Wee1 inhibition, which can be monitored through CDK2 expression and activation patterns .

• Combination therapy development: By monitoring CDK2 expression and activation in response to various therapeutic agents, researchers can identify synergistic drug combinations that overcome resistance mechanisms.

• Biomarker identification: CDK2 expression patterns detected by specific antibodies may serve as predictive biomarkers for therapeutic response or resistance in various cancer types.

• Target engagement studies: CDK2 antibodies can be used to confirm the binding of CDK inhibitors to their intended targets in both sensitive and resistant cell populations.

What are the technical considerations for multiplexed imaging studies involving CDK2 and other cell cycle markers?

Multiplexed imaging of CDK2 with other cell cycle markers requires careful technical planning:

• Antibody compatibility:

  • Select CDK2 antibodies raised in different host species than other target antibodies

  • Consider using directly conjugated primary antibodies to avoid species cross-reactivity

  • Sequential staining with complete stripping between rounds may be necessary for incompatible antibodies

• Signal separation:

  • Choose fluorophores with minimal spectral overlap

  • Include appropriate single-stained controls for spectral unmixing

  • For chromogenic multiplexing, use distinct chromogens with good separation (e.g., DAB, Fast Red, etc.)

• Subcellular localization considerations:

  • CDK2 displays both nuclear and cytoplasmic localization depending on cell type and cell cycle phase

  • Pair with markers that have distinct subcellular distributions for easier interpretation

  • Use high-resolution imaging (confocal or super-resolution) for co-localization studies

• Sample preparation:

  • Optimize fixation to preserve epitopes for all target proteins

  • Consider tissue clearing techniques for thick specimens

  • Use appropriate antigen retrieval methods compatible with all targets

• Quantification approaches:

  • Employ image analysis software capable of multi-parameter analysis

  • Quantify co-localization using established coefficients (Pearson's, Manders')

  • Consider machine learning approaches for complex pattern recognition

How might CDK2 antibodies contribute to understanding cell cycle regulation in developmental biology?

CDK2 antibodies offer significant potential for advancing developmental biology research:

• Lineage-specific cell cycle regulation: CDK2 antibodies can help characterize how cell cycle dynamics vary across different embryonic cell lineages during development.

• Tissue-specific CDK2 function: Immunohistochemistry with CDK2 antibodies can reveal tissue-specific patterns of expression during organogenesis, providing insights into specialized roles of CDK2 in different developmental contexts.

• Stem cell differentiation studies: Changes in CDK2 localization and activity during stem cell differentiation can be monitored using specific antibodies, illuminating the relationship between cell cycle regulation and cell fate determination.

• HOX gene regulation: Given that CDK2 regulates HOX genes involved in morphogenesis and differentiation , CDK2 antibodies can help elucidate the mechanistic links between cell cycle regulation and developmental patterning.

• Developmental timing mechanisms: By tracking CDK2 expression during developmental transitions, researchers can better understand how cell cycle regulation contributes to the timing of key developmental events.

What novel methodologies are emerging for studying CDK2 using antibody-based approaches?

Several innovative methodologies are enhancing the utility of CDK2 antibodies in research:

• Proximity ligation assays (PLA): This technique allows visualization of protein-protein interactions involving CDK2 in situ, enabling researchers to study CDK2-cyclin complexes and other interactions within their native cellular context.

• CRISPR-engineered endogenous tagging: Combining CRISPR gene editing with well-validated CDK2 antibodies allows tracking of endogenously tagged CDK2, providing more physiologically relevant insights than overexpression systems.

• Live-cell CDK2 activity sensors: Nanobodies derived from CDK2 antibodies coupled with fluorescent reporters enable real-time monitoring of CDK2 activity in living cells.

• Single-cell proteomics: CDK2 antibodies are being adapted for use in emerging single-cell proteomic techniques, allowing assessment of CDK2 expression heterogeneity within tissues.

• Spatial transcriptomics integration: Combining CDK2 antibody staining with spatial transcriptomics provides correlation between CDK2 protein levels and gene expression patterns within tissue architectural contexts.