CDK11A Antibody

What is CDK11A and what are its primary functions in cellular processes?

CDK11A (Cyclin-dependent kinase 11A) belongs to the CMGC Ser/Thr protein kinase family and the CDC2/CDKX subfamily, playing multiple crucial roles in cell cycle progression, cytokinesis, and apoptosis . The protein exists in different isoforms, with the p110 isoform primarily involved in pre-mRNA splicing through phosphorylation of splicing proteins such as SFRS7 . The p58 isoform functions as a negative regulator of normal cell cycle progression, suggesting its potential tumor suppressive properties . CDK11 expression is coordinately regulated with p34 (cdc2) during the cell cycle, indicating its importance in cell division control mechanisms .

Research has demonstrated that CDK11 can be cleaved by caspases during cellular stress, directly implicating it in apoptotic pathways . The gene is located near CDC2L2, a nearly identical gene in the same chromosomal region, which has created some complexity in studying its specific functions . Recent evidence suggests that CDK11 may harbor one or more tumor suppressor genes affected by chromosome 1p36 modifications in neuroblastoma, expanding its significance in cancer biology research .

What applications are CDK11A antibodies most suitable for in experimental research?

CDK11A antibodies have been validated for multiple experimental applications, making them versatile tools in molecular and cellular research. Western blotting (WB) represents one of the most common applications, with recommended dilutions typically ranging from 1:500 to 1:2000 depending on the specific antibody preparation . Immunofluorescence (IF) and immunocytochemistry (ICC) applications typically employ dilutions between 1:100 and 1:500, allowing researchers to visualize the subcellular localization of CDK11A in fixed cells .

Immunohistochemistry on paraffin-embedded tissues (IHC-P) has been validated for several commercial CDK11A antibodies, with successful staining demonstrated in tissues such as human lung carcinoma . Some antibodies have also been validated for immunoprecipitation (IP), enabling the isolation of CDK11A and its binding partners for protein interaction studies . ELISA-based applications have been confirmed for certain monoclonal antibodies, providing quantitative measurement options . When selecting the appropriate antibody, researchers should prioritize products with validation data specific to their experimental system and application to ensure optimal results.

How should researchers validate CDK11A antibody specificity before experimental use?

Proper validation of CDK11A antibody specificity is critical for ensuring reliable experimental results. Researchers should first perform Western blot analysis using positive control samples (such as COLO cell lysates or HeLa whole cell lysates) to confirm that the antibody detects proteins of the expected molecular weight—approximately 91 kDa for the primary isoform . A critical control experiment involves peptide competition assays, where pre-incubation of the antibody with the immunizing peptide should abolish specific signals, as demonstrated in validation data for certain commercial antibodies .

For immunofluorescence applications, researchers should compare staining patterns with published subcellular localization data and include negative controls omitting primary antibody to verify signal specificity . When working with tissues, comparison of staining between normal and disease samples can provide additional validation of specificity, as seen in studies showing increased nuclear intensity of CDK11 in TNBC patient tissues compared to normal tissue . Cross-validation using multiple antibodies targeting different epitopes of CDK11A can provide additional confidence in specificity, particularly when distinguishing between closely related family members like CDK11A and CDK11B .

What are the different isoforms of CDK11 and how can they be experimentally distinguished?

CDK11 exists in multiple isoforms that perform distinct cellular functions, with the two predominant forms being p110 and p58 . The p110 isoform is constitutively expressed and primarily involved in pre-mRNA splicing functions, while the p58 isoform is expressed through an internal ribosome entry site (IRES) predominantly during the G2/M phase of the cell cycle and functions as a negative regulator of cell cycle progression . These isoforms can be distinguished by their molecular weights using techniques such as Western blotting, with p110 appearing at approximately 110 kDa and p58 at 58 kDa .

From a genetic perspective, CDK11 is encoded by two highly homologous genes: CDK11A (CDC2L1) and CDK11B (CDC2L2), which share significant sequence similarity and are located in close proximity on chromosome 1p36.3 . Distinguishing between protein products of these genes requires carefully selected antibodies with validated specificity. For functional studies, researchers can employ isoform-specific siRNAs targeting unique regions of each variant, as demonstrated in breast cancer research where specific knockdown revealed distinct roles for different CDK11 forms . Additionally, the use of isoform-specific antibodies that recognize unique epitopes, such as those in the N-terminal regions where sequence variation occurs, can help differentiate between these closely related proteins .

How can CDK11A antibodies be optimized for specific cancer tissue research?

Optimizing CDK11A antibodies for cancer tissue research requires careful consideration of fixation methods, antigen retrieval techniques, and detection systems. For formalin-fixed paraffin-embedded (FFPE) cancer tissues, heat-induced epitope retrieval using citrate-based solutions (pH 6.0) has proven effective, as demonstrated in studies of lung carcinoma and breast cancer tissues . The duration of antigen retrieval should be optimized, with approximately 30 minutes being standard for many protocols . Researchers should first validate antibody performance on tissues known to express CDK11A before proceeding to experimental samples.

When working with breast cancer subtypes, particularly triple-negative breast cancer (TNBC), immunohistochemical protocols using anti-CDK11 antibodies at dilutions of 1:50 (for the Santa Cruz sc-938 antibody) have demonstrated high nuclear intensity staining compared to normal tissue . For multiplex immunofluorescence studies that examine CDK11A alongside proliferation markers like Ki-67, sequential staining protocols may be necessary to prevent cross-reactivity issues . Quantification of CDK11A expression in cancer tissues can be achieved using digital image analysis platforms, similar to the ImmunoRatio web application used for Ki-67 analysis in CDK11-related research . Background suppression using appropriate blocking reagents (such as Background Sniper in 5% skim milk) is crucial for reducing non-specific staining in cancer tissues with high protein content .

What experimental considerations are important when studying CDK11A in apoptosis pathways?

Studying CDK11A in apoptosis pathways requires special experimental design considerations, as CDK11 undergoes caspase-mediated cleavage during apoptosis, generating fragments with distinct functions . Researchers should employ time-course experiments when inducing apoptosis to capture the dynamic processing of CDK11A, collecting samples at multiple timepoints following apoptotic stimulation. Western blot analysis should utilize antibodies that can detect both full-length CDK11A and its cleavage products, with particular attention to membrane exposure times to visualize potentially faint cleavage bands.

To establish causality between CDK11A cleavage and apoptotic events, researchers should consider using caspase inhibitors (pan-caspase inhibitors or specific caspase-3 inhibitors) alongside apoptotic stimuli to determine whether preventing CDK11A cleavage affects cell death outcomes. For mechanistic studies, site-directed mutagenesis of potential caspase cleavage sites in CDK11A can be employed to generate cleavage-resistant variants for functional assessment . Co-immunoprecipitation experiments using CDK11A antibodies can help identify interaction partners that change during apoptosis, providing insights into the signaling networks involved. When performing immunofluorescence studies, dual staining with apoptotic markers (such as cleaved PARP or active caspase-3) alongside CDK11A can help correlate CDK11A localization changes with apoptotic progression in individual cells.

How can researchers address cross-reactivity issues between CDK11A and CDK11B?

Addressing cross-reactivity between CDK11A and CDK11B represents a significant challenge due to their high sequence homology. To overcome this issue, researchers should first carefully evaluate antibody specificity data, preferentially selecting antibodies raised against unique regions of each protein . Examining immunogen sequences is critical—antibodies generated against the N-terminal regions (amino acids 5-216) may provide better discrimination between CDK11A and CDK11B compared to those targeting more conserved kinase domains .

For definitive isoform-specific detection, researchers can employ complementary molecular approaches alongside immunological methods. Quantitative RT-PCR using primers specifically designed to distinguish between CDK11A and CDK11B transcripts can verify expression at the mRNA level before proceeding to protein detection . RNA interference experiments using siRNAs targeting unique regions of each gene can help validate antibody specificity by demonstrating corresponding decreases in detected protein levels . For highly sensitive applications, researchers might consider using recombinant systems expressing tagged versions of CDK11A or CDK11B as positive controls and for antibody validation. When analyzing existing literature or experimental data, careful attention should be paid to whether the antibodies used can truly distinguish between these highly similar proteins, as many commercial antibodies recognize both forms and are labeled as "CDK11A/CDK11B" antibodies .

What are best practices for quantifying CDK11A expression levels in tissue samples?

Accurate quantification of CDK11A expression in tissue samples requires standardized approaches to image acquisition and analysis. For immunohistochemistry applications, researchers should establish consistent staining protocols with appropriate positive and negative controls run in parallel with experimental samples . Digital image analysis systems should be employed for objective quantification, with algorithms that can distinguish nuclear from cytoplasmic staining, as CDK11A exhibits differential localization patterns that may have functional significance .

For semi-quantitative assessment, scoring systems based on both staining intensity (0-3+) and percentage of positive cells can be implemented, similar to those used for other nuclear markers in cancer research . When comparing expression across different tissue types or disease states, normalizing CDK11A expression to appropriate housekeeping proteins is essential for Western blot analysis, with careful selection of loading controls that remain stable across the experimental conditions . For more precise quantification, researchers can employ quantitative immunofluorescence techniques using calibrated fluorescence standards or comparing target signals to internal reference proteins within the same image. The analysis should account for potential heterogeneity within tissue samples, particularly in cancer tissues where expression may vary between tumor regions and microenvironments .

What are the recommended protocols for CDK11A antibody in immunoprecipitation studies?

For effective immunoprecipitation (IP) of CDK11A, researchers should optimize several key parameters. Cell lysis conditions should preserve protein-protein interactions while ensuring efficient extraction—typically using non-denaturing buffers containing 1% NP-40 or Triton X-100, supplemented with protease and phosphatase inhibitors to prevent degradation of CDK11A and its binding partners . Pre-clearing the lysate with protein A/G beads can help reduce non-specific binding before adding the CDK11A antibody. For the immunoprecipitation step itself, incubating 1-2 μg of CDK11A antibody with 500-1000 μg of total protein lysate overnight at 4°C provides optimal binding conditions .

Control immunoprecipitations using isotype-matched control IgG are essential to distinguish specific from non-specific interactions, as demonstrated in validation experiments with HeLa cell lysates . When analyzing CDK11A complexes, gentle washing conditions (typically 3-4 washes with lysis buffer) help maintain relevant protein interactions while removing background. For detection of co-immunoprecipitated proteins, Western blotting using antibodies against suspected interaction partners should be performed with appropriate positive controls. When studying phosphorylation-dependent interactions, phosphatase inhibitors should be included throughout the procedure, and sometimes phospho-specific antibodies may be necessary to detect modified forms of CDK11A .

How should researchers design experiments to investigate CDK11A interactions with splicing factors?

Investigating CDK11A interactions with splicing factors requires specialized experimental approaches centered on nuclear protein complexes. Researchers should begin with subcellular fractionation to isolate nuclear extracts, as the p110 isoform of CDK11A primarily functions in nuclear pre-mRNA splicing processes . Co-immunoprecipitation experiments using nuclear extracts can then be performed with antibodies against CDK11A followed by Western blotting for splicing factors like SFRS7, which has been identified as a phosphorylation target .

For direct visualization of co-localization, immunofluorescence microscopy using dual staining for CDK11A and splicing factors can be performed, with particular attention to nuclear speckle patterns where splicing components typically concentrate . Researchers might employ proximity ligation assays (PLA) to detect in situ protein-protein interactions between CDK11A and splicing factors with enhanced sensitivity. To determine the functional significance of these interactions, in vitro kinase assays using immunoprecipitated CDK11A and recombinant splicing factors as substrates can help confirm direct phosphorylation events . RNA-dependent interactions should be explored by treating samples with RNases before immunoprecipitation to distinguish direct protein-protein interactions from those mediated by RNA molecules. For comprehensive identification of CDK11A-associated splicing components, immunoprecipitation followed by mass spectrometry analysis provides an unbiased approach to mapping the complete interactome.

What controls are essential when using CDK11A antibodies in cancer research models?

When applying CDK11A antibodies in cancer research models, several critical controls must be incorporated to ensure reliable data interpretation. Positive tissue controls with known CDK11A expression patterns (such as specific breast cancer cell lines or lung carcinoma tissues) should be included alongside experimental samples in each staining batch . Negative controls using isotype-matched immunoglobulins or pre-immune serum in place of the primary antibody are essential to assess background staining levels and non-specific binding .

For studies comparing CDK11A expression between cancer and normal tissues, matched normal adjacent tissue samples provide the most appropriate control to account for patient-specific and tissue-specific variations . When evaluating antibody specificity in the context of cancer tissues, antigen competition controls where the antibody is pre-incubated with the immunizing peptide should abolish specific staining . In experiments involving CDK11A knockdown or overexpression, appropriate vector controls must be included to distinguish effects specific to CDK11A manipulation from those related to the experimental procedure itself .

For clinical correlative studies, standardized scoring systems should be established and validated by multiple independent observers to ensure reproducibility . When exploring CDK11A as a potential biomarker, receiver operating characteristic (ROC) curve analysis should be performed to determine optimal cutoff values for dichotomizing expression levels in relation to clinical outcomes. In mouse xenograft models treated with CDK11A-targeting therapies (such as siRNA-loaded nanocapsules), appropriate vehicle-treated controls and non-targeting siRNA controls are essential to distinguish specific therapeutic effects from delivery system-related effects .

How to address inconsistent staining patterns in CDK11A immunohistochemistry?

Inconsistent staining patterns in CDK11A immunohistochemistry can arise from several factors that require systematic troubleshooting. Fixation variables often contribute to inconsistency—researchers should standardize fixation protocols, ensuring consistent fixative composition, duration, and temperature . Overfixation can mask epitopes while underfixation may compromise tissue morphology. The antigen retrieval step is particularly critical; optimization experiments comparing different retrieval methods (heat-induced versus enzymatic) and buffer compositions (citrate-based at pH 6.0 versus EDTA-based at pH 9.0) should be conducted to determine optimal conditions for CDK11A detection .

Antibody concentration requires careful titration, with dilution series experiments recommended to identify the optimal antibody concentration that maximizes specific signal while minimizing background . When staining patterns appear heterogeneous within seemingly similar tissues, this may reflect biological variability in CDK11A expression rather than technical issues, particularly in cancer samples where protein expression can vary between tumor regions . To distinguish technical from biological variability, sequential sections should be stained with antibodies targeting different epitopes of CDK11A . Detection system sensitivity can also impact results—comparing different detection methods (such as polymer-based versus avidin-biotin systems) may help optimize signal intensity while maintaining specificity . For problematic tissues, extended blocking steps (30-60 minutes) with specialized blocking reagents containing both protein blockers and serum may help reduce non-specific binding .

What approaches can resolve detection issues in Western blotting with CDK11A antibodies?

Detection issues in Western blotting with CDK11A antibodies can be addressed through a systematic optimization approach. Protein extraction methods significantly impact detection—for CDK11A, which has both nuclear and cytoplasmic distribution depending on isoform and cellular context, extraction buffers should efficiently solubilize nuclear proteins, typically containing higher detergent concentrations or specialized nuclear extraction components . Sample preparation should include protease inhibitors to prevent degradation, and phosphatase inhibitors when studying phosphorylated forms of CDK11A .

For improved detection of specific isoforms, gel concentration should be optimized—8-10% polyacrylamide gels typically provide good resolution for the 91-110 kDa CDK11A proteins, while gradient gels may be preferable when detecting multiple isoforms or cleavage products simultaneously . Transfer efficiency for high molecular weight proteins can be enhanced by adding SDS (0.1%) to the transfer buffer or using lower percentage methanol. Primary antibody incubation conditions often require optimization, with overnight incubation at 4°C generally yielding better results than shorter incubations at higher temperatures .

When signal strength is suboptimal, enhanced chemiluminescence (ECL) substrate selection can make a substantial difference—high-sensitivity substrates may be required for low-abundance CDK11A detection . For tissues or cell lines with low CDK11A expression, increasing the protein loading amount or using concentration methods like immunoprecipitation before Western blotting can improve detection . If background issues persist, more stringent washing conditions (increasing salt concentration or adding 0.1% SDS to wash buffers) may help reduce non-specific binding while maintaining specific signals .

How to interpret unexpected results when studying CDK11A in different cell types?

Unexpected results when studying CDK11A across different cell types often reflect biological complexities rather than technical failures. Cell-type specific expression patterns of CDK11A isoforms should be considered—the relative abundance of p110 versus p58 isoforms can vary substantially between cell types, leading to different banding patterns in Western blots or distinct subcellular localization in immunostaining . Additionally, post-translational modifications, particularly phosphorylation states that affect CDK11A function, may differ between cell types depending on their proliferative status or differentiation state .

Alternative splicing events affecting CDK11A may generate cell-type specific variants that react differently with antibodies depending on epitope location . When unexpected expression levels are observed, researchers should verify results using multiple detection methods—combining protein detection (Western blot, immunostaining) with mRNA analysis (qRT-PCR) can help determine whether differences occur at transcriptional or post-transcriptional levels . Functional experiments using siRNA knockdown or overexpression approaches can help validate unexpected findings by demonstrating corresponding phenotypic changes .

For clinical samples, patient heterogeneity and treatment history may influence CDK11A expression and localization, particularly in cancer tissues where genetic alterations affecting chromosome 1p36 (where CDK11A is located) are common . When comparing results to published literature, careful attention should be paid to the specific antibodies used, as different antibodies may recognize distinct epitopes or isoforms of CDK11A, leading to apparently conflicting results . Integration of data from multiple experimental approaches and careful consideration of biological context are essential for proper interpretation of unexpected CDK11A results across different cell types.