CIPK4 Antibody

What is CDK4 and why is it significant in research?

CDK4 (Cyclin-dependent kinase 4) is a master regulatory protein that integrates mitogenic and oncogenic signaling with the cell division cycle. The protein has a molecular mass of approximately 33.7 kilodaltons and is encoded by a gene also known as CMM3, PSK-J3, or cell division protein kinase 4. CDK4 plays a critical role in promoting cell proliferation through interaction with D-type cyclins, facilitating the G1/S phase transition of the cell cycle. Its significance in research stems from its deregulation in most cancers, making it both an important biomarker and therapeutic target. CDK4 inhibitors have become standard treatments for metastatic estrogen-receptor positive breast cancers and are being evaluated in various other cancer types .

What types of CDK4 antibodies are available for research?

Researchers can choose from a diverse array of CDK4 antibody formats, including:

• Monoclonal antibodies: Provide high specificity for particular CDK4 epitopes

• Polyclonal antibodies: Recognize multiple epitopes of CDK4 protein

• Phospho-specific antibodies: Target specific phosphorylation sites (particularly T172)

• Tag-specific antibodies: Recognize tagged recombinant CDK4 proteins

These antibodies come in various conjugation formats (unconjugated or tagged with fluorescent dyes, HRP, etc.) and are validated for different applications including Western blotting, immunohistochemistry, immunofluorescence, flow cytometry, and ELISA .

How should I select the appropriate CDK4 antibody for my experiment?

Selecting the optimal CDK4 antibody requires consideration of multiple experimental parameters:

• Experimental application: Determine whether the antibody has been validated for your specific application (WB, IHC, IF, IP, ELISA, etc.)

• Species reactivity: Verify cross-reactivity with your experimental model (human, mouse, rat, etc.)

• Epitope specificity: For phosphorylation studies, select antibodies recognizing specific phosphorylation sites such as T172

• Clonality: Choose monoclonal antibodies for consistent results in long-term studies or polyclonal for stronger signals

• Validation evidence: Review published literature and supplier validation data including Western blot images, IF patterns, and IHC staining

• Batch consistency: For critical experiments, consider antibodies with demonstrated lot-to-lot consistency

What controls should I include when using CDK4 antibodies?

Proper experimental controls are essential for reliable interpretation of CDK4 antibody results:

• Positive controls: Include cell lines known to express CDK4 (most proliferating cell lines)

• Negative controls: Use CDK4-knockout cells or tissues, or cells treated with CDK4-specific siRNA

• Phosphorylation controls: For phospho-specific antibodies, include samples treated with phosphatase inhibitors (positive control) and phosphatases (negative control)

• Loading controls: Use housekeeping proteins (β-actin, GAPDH) to normalize CDK4 expression

• Antibody controls: Include an isotype control antibody to assess non-specific binding

• Blocking peptide controls: Use the immunizing peptide to confirm specificity in Western blots or IHC

How can I detect active CDK4 versus total CDK4 protein?

Distinguishing between active and inactive forms of CDK4 is critical for understanding its functional state in experimental systems:

• Phospho-specific antibodies: Use antibodies specifically recognizing T172-phosphorylated CDK4, which represents the active form of the kinase. The phosphorylation at T172 in the T-loop is a highly regulated step that determines cyclin D-CDK4 complex activity.

• Co-immunoprecipitation approaches:

  • Immunoprecipitate CDK4 and blot for T172 phosphorylation

  • Alternatively, use T172-phospho-specific antibodies (like LA2-AD4 mAb) for immunoprecipitation to isolate only the active CDK4 fraction

• Kinase activity assays: After immunoprecipitation of CDK4, perform in vitro kinase assays using recombinant Rb protein as substrate

• Sequential immunoprecipitation: First deplete T172-phosphorylated CDK4 using phospho-specific antibodies, then analyze the remaining fraction as inactive CDK4

What are the best methods to study CDK4-cyclin D interactions using antibodies?

CDK4-cyclin D complex analysis requires specialized approaches:

• Co-immunoprecipitation strategy:

  • Immunoprecipitate with anti-cyclin D1 or D3 antibodies and blot for CDK4

  • Alternatively, immunoprecipitate with CDK4 antibody and blot for cyclins

  • For active complexes, use phospho-T172-specific CDK4 antibodies for immunoprecipitation

• Proximity ligation assay (PLA):

  • Use paired antibodies against CDK4 and cyclin D

  • Provides spatial information about complex formation in situ

• FRET-based analysis:

  • Utilize fluorescently-labeled antibodies against CDK4 and cyclins

  • Enables live-cell analysis of complex dynamics

• Sequential immunoprecipitation to identify subcomplexes:

  • First deplete with cyclin D1 antibodies, then analyze remaining CDK4 for association with other cyclins or inhibitors

How can CDK4 antibodies predict response to CDK4/6 inhibitor therapy?

CDK4 antibodies offer valuable predictive capabilities for CDK4/6 inhibitor efficacy:

• Detection of active CDK4: T172-phosphorylated CDK4 signals the presence or absence of active CDK4 (the target of CDK4/6 inhibitory drugs) and has been associated with tumor cell sensitivity to palbociclib and other inhibitors.

• Quantitative analysis approach:

  • Use phospho-T172 specific antibodies in immunoblotting or ELISA

  • Compare phospho-CDK4 levels with total CDK4 to calculate activation ratios

  • Higher ratios correlate with increased sensitivity to CDK4/6 inhibitors

• Multiplexed analysis with pathway components:

  • Combine CDK4 antibody detection with analysis of p16, Rb, and E2F targets

  • Complete pathway profiling provides more comprehensive prediction

• Spatial context in tumors:

  • Use IHC with phospho-CDK4 antibodies to analyze heterogeneity of CDK4 activation

  • Identify regions of likely drug resistance versus sensitivity

What methods can differentiate between CDK4 and CDK6 in experimental systems?

Distinguishing these related kinases requires specific approaches:

• Antibody selection strategy:

  • Verify antibody specificity using recombinant CDK4 and CDK6 proteins

  • Select antibodies targeting non-homologous regions

  • Validate with knockout or knockdown systems for each kinase

• Sequential immunodepletion approach:

  • First deplete CDK4 using specific antibodies

  • Then analyze remaining fraction for CDK6

  • Confirm with reverse depletion sequence

• Comparative analysis workflow:

  • Run parallel reactions with CDK4-specific and CDK6-specific antibodies

  • Include control samples with known CDK4/CDK6 ratios

  • Normalize signals to create quantitative comparison

• Mass spectrometry validation:

  • Use antibodies for immunoprecipitation

  • Confirm identity by mass spectrometry

  • Identify potential cross-reactivity issues

How can I resolve weak or absent CDK4 antibody signals in Western blots?

Optimizing Western blot signals for CDK4 detection:

• Sample preparation optimization:

  • Use phosphatase inhibitors to preserve T172 phosphorylation

  • Add protease inhibitors to prevent degradation

  • Consider subcellular fractionation to enrich nuclear fraction

• Technical adjustments:

  • Increase antibody concentration or incubation time

  • Optimize blocking conditions (BSA may work better than milk for phospho-epitopes)

  • Try different membrane types (PVDF vs. nitrocellulose)

  • Increase protein loading (50-100 μg recommended for phospho-CDK4)

• Signal enhancement strategies:

  • Use more sensitive detection systems (ECL Plus, fluorescent secondary antibodies)

  • Consider antibody signal amplification systems

  • Try reducing SDS concentration in transfer buffer

• Antibody-specific factors:

  • Verify antibody storage conditions and expiration

  • Test alternative CDK4 antibody clones targeting different epitopes

  • For phospho-specific detection, compare with total CDK4 levels

What factors affect CDK4 antibody specificity in immunoprecipitation experiments?

Critical considerations for successful CDK4 immunoprecipitation:

• Buffer composition effects:

  • Use low-detergent buffers for maintaining protein-protein interactions

  • Include phosphatase inhibitors for phospho-epitope preservation

  • Adjust salt concentration based on desired stringency

• Antibody selection criteria:

  • Choose antibodies specifically validated for immunoprecipitation

  • For T172-phosphorylated CDK4, LA2-AD4 mAb (3.5 μg per 1 mg protein lysate) has been validated

  • Consider using a cocktail of antibodies recognizing different epitopes

• Technical optimization parameters:

  • Adjust antibody-to-lysate ratio (typically 3-5 μg antibody per mg of protein)

  • Optimize incubation time and temperature (overnight at 4°C generally recommended)

  • Pre-clear lysates to reduce non-specific binding

• Validation approaches:

  • Confirm specificity by Western blotting of immunoprecipitated material

  • Include IgG control to identify non-specific binding

  • Consider reverse immunoprecipitation with known interacting partners

How can I develop and validate phospho-specific CDK4 antibodies?

Methodological approach for phospho-CDK4 antibody development:

• Immunogen design strategy:

  • Use long phosphopeptides that include the complete activation segment of CDK4

  • For T172 phosphorylation, design peptides with phosphothreonine residue centered

  • Consider KLH fusion for improved immunogenicity

• Screening protocol:

  • Develop ELISA screening with both phosphorylated and non-phosphorylated peptides

  • Create phosphorylated and non-phosphorylated cyclin D/CDK4 fusions with biotinylatable tags for validation

  • Screen hybridoma supernatants for differential binding

• Validation sequence:

  • Test against recombinant phosphorylated versus non-phosphorylated proteins

  • Validate with lysates from cells treated with or without CDK4 activators

  • Confirm with CDK4 knockout or knockdown controls

  • Test sensitivity to phosphatase treatment

• Cross-reactivity assessment:

  • Check for reactivity against similar phosphorylation sites in related CDKs

  • Validate specificity across species (human, mouse, etc.)

What methodologies can detect CDK4 interaction with CDK inhibitors (CKIs) like p21 and p27?

Specialized techniques for studying CDK4-CKI interactions:

• Co-immunoprecipitation workflow:

  • Immunoprecipitate with T172-phosphorylated CDK4-specific antibodies

  • Analyze co-precipitated p21 and p27 with phospho-specific antibodies

  • Research indicates privileged interaction of T172-phosphorylated CDK4 with S130-phosphorylated p21 and S10-phosphorylated p27

• Sequential immunoprecipitation approach:

  • First immunoprecipitate with CDK4 antibodies

  • Then analyze bound fraction for CKIs

  • Re-immunoprecipitate remaining lysate to quantify unbound CKIs

• In situ analysis methods:

  • Use proximity ligation assays to visualize CDK4-CKI interactions

  • Perform FRET analysis with labeled antibodies to study interaction dynamics

• Quantitative analysis strategy:

  • Develop ELISA systems using capture antibodies against CDK4

  • Detect bound CKIs with specific detection antibodies

  • Compare ratios of CKI-bound versus free CDK4

How can computational modeling enhance CDK4 antibody specificity?

Advanced computational approaches for antibody engineering:

• Biophysics-informed modeling approach:

  • Train models on experimentally selected antibodies

  • Associate each potential ligand with a distinct binding mode

  • Use this to predict and generate specific variants beyond experimental observations

• Phage display optimization workflow:

  • Conduct phage display experiments with antibody selection against diverse ligand combinations

  • Use high-throughput sequencing to analyze selected antibodies

  • Feed data into computational models to identify binding determinants

• Custom specificity profile design:

  • Generate cross-specific sequences by jointly minimizing energy functions associated with desired ligands

  • Create specific sequences by minimizing energy functions for desired ligands while maximizing them for undesired ones

  • Validate computationally designed antibodies experimentally

What single-cell approaches can utilize CDK4 antibodies for cell cycle research?

Cutting-edge single-cell methodologies:

• Multiplexed imaging technique:

  • Combine CDK4 phospho-antibodies with markers for cell cycle phases

  • Use cyclin antibodies to create comprehensive cell cycle profiles

  • Apply spectral unmixing for simultaneous detection of multiple markers

• Flow cytometry-based approach:

  • Pair phospho-CDK4 antibodies with DNA content analysis

  • Add EdU incorporation to identify S-phase cells

  • Create multidimensional datasets for cell cycle progression analysis

• Mass cytometry (CyTOF) strategy:

  • Use metal-tagged CDK4 antibodies with other cell cycle markers

  • Analyze dozens of parameters simultaneously

  • Quantify CDK4 activity across heterogeneous cell populations

• Single-cell Western blot application:

  • Analyze CDK4 phosphorylation in individual cells

  • Correlate with expression of other cell cycle regulators

  • Identify rare cell populations with unique CDK4 activation profiles