CDK6 Antibody

What is CDK6 and why is it an important research target?

CDK6 is a serine/threonine-protein kinase that plays critical roles in cell cycle regulation and cellular differentiation. It primarily promotes G1/S transition by phosphorylating key proteins like pRB/RB1 and NPM1, and interacts with D-type G1 cyclins to form pRB/RB1 kinase complexes that control cell cycle entry . Beyond its canonical role in cell cycle regulation, CDK6 is involved in the maintenance of specific cell populations, including erythroid and hematopoietic cells, and is essential for proliferation within the dentate gyrus of the hippocampus . The importance of CDK6 as a research target stems from its dual functions in both promoting and inhibiting cell proliferation depending on cellular context, and its involvement in critical signaling pathways that influence cancer development and progression .

What are the main applications for CDK6 antibodies in research?

CDK6 antibodies serve multiple experimental applications in research laboratories:

These applications enable researchers to study CDK6 expression levels, subcellular localization, protein interactions, and post-translational modifications in various experimental contexts . When selecting application parameters, it's crucial to optimize conditions for each specific cell line or tissue type being examined.

How do I select the appropriate CDK6 antibody for my experiment?

Selecting the appropriate CDK6 antibody requires consideration of several key factors:

• Antibody type: Monoclonal antibodies (like clone 8H4) provide high specificity for particular epitopes, while polyclonal antibodies may offer better sensitivity but potential cross-reactivity .

• Host species: Consider the host species (rabbit, mouse) in relation to your experimental design, particularly for multicolor immunofluorescence where avoiding primary antibody host conflicts is essential .

• Validation status: Prioritize antibodies validated in knockout/knockdown systems that demonstrate specificity for the CDK6 protein .

• Target region: Some antibodies target specific regions of CDK6 (e.g., N-terminal aa 1-100), which may affect detection of splice variants or modified forms .

• Required applications: Verify that the antibody has been validated for your specific application (WB, IHC, IF, FC, IP) with published validation data .

Before proceeding with large-scale experiments, conduct preliminary validation in your specific biological system to confirm specificity and optimize working conditions.

What controls should I include when using CDK6 antibodies?

Proper controls are essential for generating reliable data with CDK6 antibodies:

• Positive controls: Include cell lines known to express CDK6 such as HeLa, Jurkat, HepG2, K-562, or HEK-293 cells, which have been validated for many commercial CDK6 antibodies .

• Negative controls: Utilize CDK6-knockout or CDK6-knockdown cells where available; alternatively, use cells known to express minimal CDK6 .

• Isotype controls: Include matched isotype control antibodies (same species, isotype, and concentration) to assess non-specific binding, particularly important for flow cytometry and immunohistochemistry .

• Loading controls: For western blotting, include housekeeping proteins (β-actin, GAPDH) to normalize CDK6 expression levels between samples.

• Peptide competition: Consider performing peptide competition assays where the antibody is pre-incubated with the immunizing peptide to confirm binding specificity.

These controls help distinguish genuine CDK6 detection from technical artifacts and enable proper interpretation of experimental results.

Why might CDK6 antibody staining show inconsistent results between applications?

Inconsistent CDK6 antibody staining can stem from several technical factors:

• Epitope accessibility: Different sample preparation methods may alter protein conformation and epitope accessibility. For instance, formalin fixation can mask epitopes that are readily detected in frozen samples . For IHC applications with inconsistent results, try multiple antigen retrieval methods - both TE buffer pH 9.0 and citrate buffer pH 6.0 have been reported effective for CDK6 detection .

• Expression level variations: CDK6 expression fluctuates throughout the cell cycle and varies between cell types. Synchronize cells when appropriate to reduce this variability.

• Post-translational modifications: CDK6 undergoes phosphorylation and other modifications that may affect antibody recognition. Some antibodies may preferentially detect specific modified forms .

• Isoform specificity: Ensure your antibody recognizes all relevant CDK6 isoforms in your biological system.

• Cross-reactivity: Some antibodies may cross-react with structurally similar proteins, particularly CDK4. Validate specificity with siRNA knockdown experiments targeting CDK6 specifically .

When troubleshooting inconsistent results, systematically adjust sample preparation, antibody concentration, incubation conditions, and detection methods while maintaining appropriate controls.

How can I optimize CDK6 antibody performance for immunohistochemistry in different tissue types?

Optimizing CDK6 antibody performance for immunohistochemistry requires tissue-specific adjustments:

• Fixation optimization: While formalin-fixed paraffin-embedded (FFPE) tissues are standard, overfixation can mask CDK6 epitopes. Consider fixation time optimization or alternative fixatives for sensitive applications.

• Antigen retrieval customization:

  • For epithelial tissues: TE buffer (pH 9.0) often provides optimal results

  • For lymphoid tissues: Citrate buffer (pH 6.0) may be more effective

  • For difficult tissues: Consider dual pH antigen retrieval methods or enzymatic approaches

• Signal amplification: For tissues with low CDK6 expression, employ signal amplification systems such as polymer-based detection or tyramide signal amplification.

• Blocking optimization: Adjust blocking solutions based on tissue type; lymphoid tissues often require stronger blocking to prevent non-specific binding.

• Scoring system standardization: Implement a consistent scoring system based on both staining intensity (0-3) and percentage of positive cells to enable comparison across experiments, as described in recent CDK6 studies .

When working with specific cancers (lung cancer, gliomas, endometrial cancer), consult recent literature for tissue-specific protocols, as these have been validated for CDK6 detection .

What are the best approaches for quantifying CDK6 expression levels?

Accurate quantification of CDK6 expression requires application-specific approaches:

• Western blot quantification:

  • Use gradient gels (10-12%) to resolve the 36-40 kDa CDK6 band

  • Include recombinant CDK6 standards for absolute quantification

  • Analyze band intensity with software like ImageJ, normalizing to loading controls

  • For accuracy, maintain samples within the linear range of detection

• Immunohistochemistry scoring:

  • Implement the formula: staining score = percentage score × intensity score

  • Use standardized intensity scales: 0 (negative), 1 (weak/light brown), 2 (moderate/brown), 3 (strong/dark brown)

  • Establish cut-off values based on median scores from large cohorts (e.g., 27.931 in recent studies)

  • Have two independent pathologists score to reduce subjectivity

• Flow cytometry quantification:

  • Use fluorescence quantitation beads to establish standard curves

  • Report data as molecules of equivalent soluble fluorochrome (MESF)

  • Analyze median fluorescence intensity rather than mean values for robust results

• RT-qPCR for mRNA levels:

  • Design primers specific to CDK6 that avoid homologous regions with CDK4

  • Use multiple reference genes for normalization

  • Consider analyzing splice variants separately

Each approach provides complementary information, with protein-based methods (WB, IHC, flow cytometry) directly measuring the functional protein, while RT-qPCR provides insights into transcriptional regulation.

How can CDK6 antibodies be used to investigate the non-canonical functions of CDK6?

Investigating CDK6's non-canonical functions requires specialized approaches with CDK6 antibodies:

• Chromatin immunoprecipitation (ChIP):

  • Use CDK6 antibodies to identify genomic regions directly bound by CDK6, revealing its role as a transcriptional regulator

  • Combine with sequencing (ChIP-seq) to map genome-wide CDK6 binding sites

  • This approach has revealed CDK6 regulation of genes like p16^INK4a and VEGF-A independent of its kinase activity

• Co-immunoprecipitation for protein interaction networks:

  • Precipitate CDK6 complexes using specific antibodies (0.5-4.0 μg per 1-3 mg lysate)

  • Identify interaction partners through mass spectrometry

  • This technique has identified CDK6 interactions with transcription factors and components of the GSK3β/Wnt/β-catenin pathway

• Proximity ligation assays:

  • Visualize and quantify CDK6 interactions with specific partners in situ

  • Particularly useful for studying context-dependent interactions in different cell compartments

• Immunofluorescence co-localization:

  • Determine subcellular localization of CDK6 in relation to potential interaction partners

  • Use confocal microscopy to verify nuclear translocation during transcriptional activities

• Kinase-dead mutant studies:

  • Compare antibody-detected CDK6 functions between wild-type and kinase-dead mutants to distinguish between catalytic and scaffolding roles

These approaches have revealed CDK6's dual roles in both promoting and inhibiting cell proliferation, depending on p16^INK4a status, and its involvement in promoting angiogenesis through transcriptional activities independent of its kinase function .

How do CDK6 antibodies help elucidate CDK6's role in cancer progression and drug resistance?

CDK6 antibodies provide crucial tools for investigating cancer progression and drug resistance mechanisms:

• Tissue microarray analysis:

  • CDK6 antibodies enable systematic screening of tumor samples across cancer types

  • Recent studies using IHC have revealed CDK6 upregulation as a biomarker for acquired lenvatinib resistance in hepatocellular carcinoma

  • Standardized scoring systems allow correlation of CDK6 expression with patient outcomes

• Signaling pathway elucidation:

  • Multiplex immunostaining combining CDK6 antibodies with pathway markers reveals regulatory mechanisms

  • This approach identified the ERK/YAP1 signaling cascade as a mediator of CDK6 upregulation in treatment-resistant tumors

  • Co-staining with GSK3β and β-catenin helped discover non-canonical CDK6 functions in Wnt signaling activation

• Drug sensitivity prediction:

  • CDK6 expression profiling with antibodies helps predict response to CDK4/6 inhibitors like palbociclib

  • The correlation between CDK6 IHC scores and treatment efficacy informs personalized medicine approaches

• Tumor immune microenvironment characterization:

  • Combined analysis of CDK6 with immune markers reveals its impact on tumor immunology

  • High CDK6 expression correlates with distinct immune cell infiltration patterns, including reduced resting memory CD4+ T cells and increased M0 macrophages

  • Expression of effector molecules like GZMH, GZMA, GZMB, GZMM, IFNG, and PRF1 is enriched in high-CDK6 contexts

• Therapeutic resistance mechanisms:

  • Sequential sampling and CDK6 antibody staining during treatment reveals dynamic changes driving resistance

  • CDK6 upregulation has been identified as a druggable target in lenvatinib-resistant HCC, with combined CDK6 inhibition showing synergistic effects

These applications demonstrate how CDK6 antibodies contribute to understanding cancer biology beyond simple expression analysis, revealing mechanistic insights with therapeutic implications.

What approaches can resolve discrepancies between CDK6 mRNA and protein expression data?

Researchers frequently encounter discrepancies between CDK6 mRNA and protein levels. Resolving these discrepancies requires multilevel analytical approaches:

• Temporal analysis:

  • CDK6 mRNA and protein may have different half-lives and temporal dynamics

  • Design time-course experiments sampling both mRNA (RT-qPCR) and protein (Western blot) at multiple timepoints

  • This approach revealed that in MLL-rearranged ALL, CDK6 mRNA overactivation precedes protein upregulation

• Post-transcriptional regulation assessment:

  • Investigate microRNA regulation of CDK6 using anti-AGO2 RIP-seq

  • Analyze polysome profiling to determine translational efficiency of CDK6 mRNA

  • Compare cytoplasmic vs. total mRNA levels to identify export/localization issues

• Protein stability determination:

  • Use cycloheximide chase assays with CDK6 antibodies to measure protein half-life

  • Compare proteasome inhibition effects on CDK6 protein levels versus mRNA

  • Investigate post-translational modifications that affect stability using modification-specific antibodies

• Single-cell analysis:

  • Combine single-cell RNA-seq with CyTOF or single-cell Western approaches

  • This reveals population heterogeneity that may mask correlations in bulk analyses

  • CDK6 often shows bimodal expression patterns across cancer cell populations

• Methodological validation:

  • Ensure antibodies detect all relevant CDK6 isoforms

  • Verify primer specificity to exclude amplification of homologous sequences

  • Use multiple antibodies targeting different CDK6 epitopes to confirm protein quantification

A comprehensive approach combining these methods provides mechanistic insights into the regulation of CDK6 expression at multiple levels, explaining apparent discrepancies between mRNA and protein data in experimental and clinical samples.

How can CDK6 antibodies help distinguish between the roles of CDK4 versus CDK6 in specific cellular contexts?

Distinguishing between CDK4 and CDK6 functions requires careful experimental design with highly specific antibodies:

• Validation of antibody specificity:

  • Perform side-by-side testing in CDK4-knockout and CDK6-knockout cell lines

  • Use competitive binding assays with recombinant CDK4 and CDK6 proteins

  • Select antibodies targeting non-conserved regions between these homologous kinases

• Sequential immunoprecipitation approach:

  • First immunoprecipitate with CDK6-specific antibody, then probe depleted lysate with CDK4 antibody

  • This technique separates CDK6-specific complexes from CDK4 complexes

  • Helps identify unique binding partners for each kinase

• Combinatorial knockdown experiments:

  • Compare phenotypes after CDK4-specific, CDK6-specific, and combined knockdowns

  • Use validated antibodies to confirm protein depletion

  • This approach revealed that CDK6, but not CDK4, is required for proliferation of MLL-ALL cells

• Substrate-specific phosphorylation analysis:

  • Use phospho-specific antibodies for known substrates after specific kinase inhibition

  • Identify differential phosphorylation patterns between CDK4 and CDK6

  • Combined with mass spectrometry to discover novel substrate specificity

• Subcellular localization comparison:

  • Perform fractionation followed by Western blot or immunofluorescence

  • Identify differential localization patterns between CDK4 and CDK6

  • CDK6 shows distinct nuclear functions in transcriptional regulation not shared by CDK4

These approaches have revealed significant functional differences, including CDK6's unique role in transcriptional regulation unrelated to its kinase activity and its specific importance in certain cancer contexts like MLL-rearranged leukemias.

How are CDK6 antibodies being used to develop new therapeutic approaches for cancer?

CDK6 antibodies are instrumental in developing novel therapeutic strategies:

• Biomarker-driven patient stratification:

  • IHC scoring systems using CDK6 antibodies help identify patients likely to respond to CDK4/6 inhibitors

  • Cut-off values (e.g., staining score of 27.931) stratify patients into high and low CDK6 expression groups

  • This approach is particularly important for precision medicine in breast cancer, leukemia, and HCC

• Combination therapy development:

  • CDK6 antibody screening identifies tumors with dual pathway activation

  • In HCC, CDK6 upregulation mediates lenvatinib resistance, and CDK6 inhibition with palbociclib shows synergistic effects with lenvatinib

  • Antibody-based screening reveals optimal timing for sequential or combination treatments

• Proteolysis targeting chimera (PROTAC) development:

  • CDK6 antibodies evaluate the efficacy of CDK6-targeting PROTACs

  • Unlike kinase inhibitors, PROTACs degrade the entire protein, eliminating both kinase-dependent and kinase-independent functions

  • This approach is particularly valuable for targeting CDK6's transcriptional activities that promote angiogenesis

• Immune microenvironment modulation:

  • High CDK6 expression correlates with distinct immune cell infiltration patterns

  • CDK6 inhibition reshapes the tumor immune microenvironment, potentially enhancing immunotherapy efficacy

  • Antibody-based multiplex imaging identifies optimal immunotherapy combinations

• Development of kinase-independent function inhibitors:

  • Antibodies help identify compounds that disrupt CDK6's scaffolding and transcriptional functions

  • This represents a novel therapeutic approach distinct from traditional kinase inhibition

These applications demonstrate how CDK6 antibodies contribute not only to understanding basic biology but also to translating these insights into clinically relevant therapeutic strategies.

What are the most reliable methods to assess CDK6 antibody specificity and validate experimental results?

Ensuring CDK6 antibody specificity requires comprehensive validation:

• Genetic validation approaches:

  • CRISPR/Cas9 knockout of CDK6 provides the gold standard negative control

  • siRNA knockdown demonstrates specificity with reduced expression

  • Rescue experiments with CDK6 overexpression confirm specificity of observed effects

• Multi-antibody concordance testing:

  • Use multiple antibodies targeting different CDK6 epitopes

  • Agreement between antibodies in various applications increases confidence in specificity

  • Discordant results suggest epitope-specific detection or potential cross-reactivity

• Cross-species reactivity assessment:

  • Compare antibody performance in human, mouse, and rat samples

  • Evolutionary conservation of the target epitope suggests functional importance

  • CDK6 antibody 19117-1-AP shows validated reactivity across these species

• Application-specific controls:

  • For IHC: Include isotype controls and peptide competition assays

  • For WB: Include molecular weight markers and positive control lysates (HeLa, Jurkat cells)

  • For IP: Perform reverse IP and confirm interaction partners with alternative methods

• Orthogonal technique validation:

  • Verify key findings with non-antibody-based techniques

  • Compare protein detection with mRNA analysis (qPCR, RNA-seq)

  • Validate functional studies with genetic approaches (knockout/knockdown)

Rigorous validation is particularly important when investigating CDK6's non-canonical functions or when distinguishing between CDK4 and CDK6 due to their structural similarity.

How can CDK6 antibodies facilitate the investigation of CDK6's role in the tumor immune microenvironment?

CDK6 antibodies enable multifaceted analysis of CDK6's impact on tumor immunity:

• Multiplex immunofluorescence profiling:

  • Combine CDK6 antibodies with immune cell markers (CD8, CD4, CD68)

  • Spatial analysis reveals associations between CDK6 expression and immune cell localization

  • This approach identified correlations between high CDK6 expression and reduced resting memory CD4+ T cells with increased M0 macrophages

• Single-cell analysis of tumor ecosystems:

  • Flow cytometry with CDK6 antibodies enables sorting of tumor subpopulations

  • Subsequent transcriptomic analysis reveals differential immune regulation

  • Identifies cell-intrinsic mechanisms by which CDK6 modulates immune signaling

• Cytokine production assessment:

  • Correlate CDK6 expression with cytokine profiles in tumor microenvironments

  • GO and KEGG analyses have linked CDK6 expression to "Cytokine-cytokine receptor interaction" pathways

  • Immune effector molecule expression (GZMH, GZMA, GZMB, GZMM, IFNG, PRF1) correlates with CDK6 levels

• Checkpoint molecule expression correlation:

  • Use CDK6 antibodies alongside checkpoint molecule staining (PD-L1, PD-1, CTLA-4)

  • Determine whether CDK6 expression predicts immunotherapy response

  • This information guides combination therapy approaches with CDK4/6 inhibitors and immune checkpoint blockers

• Intervention studies with immune readouts:

  • Assess changes in immune infiltration after CDK6 inhibition

  • CDK6 inhibitors like palbociclib have been shown to reshape the tumor immune microenvironment

  • Sequential biopsies with CDK6 and immune marker staining track treatment-induced changes

These techniques reveal CDK6's previously underappreciated role in modulating tumor immunity, with implications for combining targeted and immunotherapeutic approaches.

What are the key considerations for reproducible CDK6 antibody-based research?

Reproducible CDK6 antibody research depends on several critical factors:

• Detailed reporting of antibody characteristics:

  • Always report catalog numbers, clone names, and lot numbers

  • Include validation methods used to confirm specificity

  • Document dilutions, incubation conditions, and detection systems

• Consistent scoring and quantification:

  • Implement standardized scoring systems for IHC (intensity × percentage)

  • Use digital image analysis when possible to reduce subjectivity

  • Report cut-off determination methods transparently (e.g., median value from distribution)

• Comprehensive controls:

  • Include technical controls (isotype, secondary-only)

  • Incorporate biological controls (positive, negative, knockdown)

  • Report control data alongside experimental results

• Context-specific optimization:

  • Recognize that optimal conditions vary between tissue types

  • Document tissue-specific modifications to protocols

  • Report antigen retrieval methods used (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Data sharing and protocol transparency:

  • Share detailed protocols via repositories (protocols.io)

  • Submit unprocessed images to data repositories

  • Make analysis code available when using computational image analysis

Adherence to these practices enhances reproducibility and accelerates collective knowledge advancement in CDK6 research.

What emerging techniques are enhancing the utility of CDK6 antibodies in cancer research?

Innovative technologies are expanding CDK6 antibody applications:

• Spatial transcriptomics integration:

  • Combining CDK6 antibody staining with spatial transcriptomics

  • This reveals relationships between CDK6 protein expression and local transcriptional programs

  • Particularly valuable for understanding CDK6's role as a transcriptional regulator in its microenvironment

• Live-cell CDK6 dynamics:

  • Cell-permeable fluorescently labeled CDK6 antibody fragments

  • These enable real-time monitoring of CDK6 localization during cell cycle progression

  • Reveal previously unappreciated dynamic behaviors of CDK6 in living cells

• High-content screening platforms:

  • Automated CDK6 antibody-based screening in drug discovery

  • Identify compounds that modulate CDK6 expression, localization, or interaction networks

  • Screen for drugs that specifically disrupt CDK6's non-canonical functions

• Antibody-based proximity labeling:

  • CDK6 antibodies conjugated to enzymes like APEX2 or TurboID

  • These identify proteins in close proximity to CDK6 in specific cellular compartments

  • Reveal context-specific interaction networks difficult to capture with traditional co-IP

• Single-cell proteomics integration:

  • CDK6 antibodies in CyTOF and CODEX multiplexed imaging

  • These approaches reveal heterogeneity in CDK6 expression and function at single-cell resolution

  • Particularly important for understanding resistance mechanisms in complex tumors

These emerging techniques promise to deepen our understanding of CDK6 biology and accelerate translation into therapeutic approaches for cancer and other diseases.

How can researchers integrate CDK6 antibody data with other -omics approaches for comprehensive insights?

Multi-omics integration with CDK6 antibody data yields comprehensive insights:

• Integrated proteogenomic analysis:

  • Correlate CDK6 protein levels (antibody-based) with genomic alterations

  • Identify mechanisms driving discordance between CDK6 genomic, transcriptomic, and proteomic data

  • This approach revealed that while CDK6 copy number alterations correlate with mRNA levels, protein abundance is often regulated post-transcriptionally

• Phospho-proteomic correlation:

  • Combine CDK6 antibody-based expression data with phospho-proteomics

  • Map CDK6-dependent phosphorylation networks

  • Identify novel substrates and signaling pathways affected by CDK6 modulation

• Multi-modal single-cell analysis:

  • Integrate CDK6 protein detection with single-cell transcriptomics

  • This reveals cell state-specific relationships between CDK6 expression and gene programs

  • Particularly valuable for understanding heterogeneous responses to CDK4/6 inhibitors

• Metabolomic integration:

  • Correlate CDK6 protein levels with metabolic profiles

  • Investigate how CDK6 influences cellular metabolism beyond cell cycle regulation

  • This approach has revealed connections between CDK6 and metabolic reprogramming in cancer

• Clinical data integration:

  • Combine CDK6 IHC scoring with patient genomic, transcriptomic, and clinical data

  • Develop multiparameter predictive models for treatment response

  • Recent studies have shown that integrating CDK6 protein data with transcriptomic signatures improves prediction of CDK4/6 inhibitor efficacy compared to either alone