CDK5 Monoclonal Antibody

What is CDK5 and why is it important in research?

Cyclin-dependent kinase 5 (CDK5) is a unique member of the cyclin-dependent kinase family that, unlike its counterparts, is not primarily involved in cell cycle regulation but instead plays crucial roles in neuronal development, migration, and synaptic function. CDK5 is tightly regulated through association with activator proteins p35 and p39, which can be cleaved by calpain to form p25 and p29, leading to prolonged activation of CDK5 and potentially pathological consequences . Research has revealed CDK5's involvement in neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis (ALS), where accumulation of p25 has been observed . More recent studies have also established CDK5 as a regulator of the circadian clock, specifically involved in the nuclear import of Period 2 (PER2), a key circadian clock component . CDK5's diverse functions extend beyond the nervous system, with emerging roles in cancer progression, pain sensation, and various other cellular activities, making it a critical target for research across multiple disciplines .

How do monoclonal CDK5 antibodies differ from polyclonal ones?

Monoclonal CDK5 antibodies are derived from a single B-cell clone, providing high specificity for a single epitope of the CDK5 protein, which results in consistent and reproducible experimental outcomes. These antibodies typically exhibit lower background signal and cross-reactivity compared to polyclonal alternatives, making them ideal for applications requiring precise detection of CDK5 without interference from similar proteins such as CDK1-4 . Polyclonal CDK5 antibodies, in contrast, are produced from multiple B-cell lineages and recognize multiple epitopes on the CDK5 protein, potentially offering higher sensitivity but with greater batch-to-batch variation and possible cross-reactivity. Most commercially available monoclonal CDK5 antibodies are generated using specific recombinant fragments of human CDK5 expressed in E. coli as immunogens, as seen with antibody clone 4E4, which recognizes a specific portion of human CDK5 . The choice between monoclonal and polyclonal antibodies depends on the research application, with monoclonals generally preferred for quantitative analyses and applications where distinguishing between closely related proteins is crucial.

What should researchers consider when selecting a CDK5 monoclonal antibody?

Researchers should first determine the specific application requirements, as different CDK5 antibodies are optimized for particular techniques such as Western blotting, immunoprecipitation, immunohistochemistry, or flow cytometry. The species reactivity is a critical consideration, with available antibodies showing different cross-reactivity patterns with human, mouse, rat, and monkey CDK5 proteins . For instance, some antibodies react only with human CDK5, while others recognize the protein across multiple species, which is essential for comparative or translational studies . Epitope specificity must be carefully evaluated, as antibodies targeting different regions of CDK5 (N-terminal, central region, or C-terminal) may yield different results depending on protein conformation, post-translational modifications, or protein-protein interactions. Antibodies recognizing specific phosphorylation sites, such as Tyr15, provide valuable information about CDK5's activation state, although it's worth noting that Tyr15 phosphorylation appears to occur primarily on inactive monomeric CDK5 . The clonality and isotype of the antibody (such as IgG1 for the 4E4 clone) affect compatibility with secondary detection systems and potential for multiplexing with other antibodies .

What are the recommended protocols for using CDK5 monoclonal antibodies in Western blotting?

For Western blotting applications, CDK5 monoclonal antibodies should be used at dilutions ranging from 1:500 to 1:5000, with most manufacturers recommending around 1:1000 for optimal results . Researchers should prepare protein lysates using lysis buffers that preserve phosphorylation states if studying CDK5 regulation, and typical sample loading should range from 20-50 μg of total protein per lane. When resolving CDK5 by SDS-PAGE, a 30 kDa band should be expected, which helps distinguish it from related CDKs of different molecular weights . Membrane transfer conditions should be optimized for proteins in this size range, typically using PVDF membranes and standard transfer buffers. Blocking should be performed with 5% non-fat dry milk or bovine serum albumin in TBST, with the latter preferred when detecting phosphorylated forms of CDK5. Incubation with primary CDK5 antibody should occur overnight at 4°C or for 2 hours at room temperature, followed by appropriate secondary antibody incubation and washing steps. Importantly, researchers should include positive controls (known CDK5-expressing samples) and negative controls (samples from CDK5 knockout models or CDK5-silenced cells) to validate specificity .

How can researchers effectively use CDK5 antibodies for immunoprecipitation?

For immunoprecipitation of CDK5, researchers should use antibodies specifically validated for this application, such as those recommended at a 1:50 dilution . A detailed protocol involves preparing cell or tissue lysates in a non-denaturing buffer that preserves protein-protein interactions, particularly those between CDK5 and its activators p35/p39. Pre-clearing the lysate with Protein A/G beads helps reduce non-specific binding before adding the CDK5 antibody at the recommended concentration. The antibody-lysate mixture should be incubated overnight at 4°C with gentle rotation, followed by addition of Protein A/G beads for antibody capture . Using magnetic Dynabeads can improve recovery and reduce background compared to traditional agarose beads. After thorough washing to remove non-specifically bound proteins, the immunoprecipitated CDK5 complex can be eluted for downstream applications such as kinase assays or mass spectrometry analysis. A critical control is the parallel immunoprecipitation using isotype-matched non-specific IgG antibodies such as AffiniPure Goat Anti-Rabbit IgG or AffiniPure Goat Anti-Mouse IgG, depending on the host species of the CDK5 antibody . For co-immunoprecipitation studies investigating CDK5 interactions with binding partners, gentler wash conditions may be necessary to preserve these interactions.

What considerations should be made when using CDK5 antibodies for immunohistochemistry and immunofluorescence?

For immunohistochemistry (IHC) and immunofluorescence (IF) applications, CDK5 monoclonal antibodies should be used at dilutions ranging from 1:20 to 1:200, with optimization for each specific tissue type and fixation method . Proper fixation is crucial, with 4% paraformaldehyde typically providing the best results for preserving CDK5 epitopes while maintaining tissue morphology. Antigen retrieval methods should be carefully selected and optimized, as CDK5 epitopes may be masked during fixation. When performing double or triple immunostaining, researchers should ensure compatibility between the CDK5 antibody and other primary antibodies in terms of host species and isotype to prevent cross-reactivity of secondary antibodies. CDK5 typically shows both cytoplasmic and nuclear localization, with the distribution pattern potentially changing under different physiological or pathological conditions, so appropriate nuclear counterstaining (such as DAPI) is recommended for accurate interpretation of results . Well-designed controls are essential, including tissue from CDK5-silenced animals as negative controls, as demonstrated in studies using shCDK5 in the suprachiasmatic nucleus (SCN) where efficient knockdown of CDK5 was confirmed by the absence of staining with anti-CDK5 antibodies compared to scrambled shRNA controls .

What protocols can be used to measure CDK5 kinase activity?

Measuring CDK5 kinase activity requires a combination of immunoprecipitation and kinase assay techniques, with both radioactive and non-radioactive options available to researchers. The standard protocol begins with immunoprecipitation of CDK5 from protein lysates using specific anti-CDK5 antibodies bound to Protein A/G beads or magnetic Dynabeads . The immunoprecipitated CDK5, which may still be complexed with its activators p35 or p39 (or their calpain-cleaved derivatives p25 or p29), is then used in a kinase reaction with an appropriate substrate such as histone H1 or a CDK5-specific peptide substrate . In the radioactive approach, the kinase reaction includes [γ-32P]ATP, and CDK5 activity is measured by the incorporation of 32P into the substrate, quantified by scintillation counting or phosphorimaging. Non-radioactive alternatives include using ATP-γS followed by detection with a thiophosphate ester antibody, or employing phospho-specific antibodies that recognize the phosphorylated form of the substrate. A crucial control is the parallel immunoprecipitation using non-specific IgG matched to the host species of the CDK5 antibody, which establishes the background level of kinase activity . For more accurate results, researchers should normalize the kinase activity to the amount of CDK5 protein recovered in the immunoprecipitation, as determined by Western blotting.

How can researchers distinguish between CDK5 activity and other CDKs in their experiments?

Distinguishing CDK5 activity from that of other CDKs requires careful selection of antibodies, substrates, and inhibitors, given the structural similarities and overlapping substrate specificities among CDK family members. Researchers should first employ highly specific CDK5 antibodies that have been validated to not cross-react with other CDKs, such as antibodies that detect endogenous levels of total CDK5, but not CDK1-4 proteins . Using immunoprecipitation with CDK5-specific antibodies before kinase assays can physically separate CDK5 from other CDKs, reducing potential contamination from similar kinases. CDK5 has unique activators (p35 and p39) distinct from the cyclins that activate other CDKs, so co-immunoprecipitation and detection of these specific activators can provide additional confirmation of CDK5-specific activity . When selecting inhibitors for in vitro or cellular studies, researchers should consider the differential sensitivities of CDK family members to various inhibitors, using the most selective available for CDK5. Additionally, conducting parallel experiments in systems where CDK5 has been genetically knocked down or knocked out provides a powerful negative control, as demonstrated in studies using shCdk5 in mice where CDK5 expression was efficiently suppressed in the suprachiasmatic nucleus .

What are the key factors affecting CDK5 activity in different tissues and experimental conditions?

CDK5 activity is regulated by multiple factors that researchers must consider when designing experiments across different tissues and conditions. The expression levels and activation state of CDK5 regulators, particularly p35 and p39, are primary determinants of kinase activity, with the calpain-cleaved forms p25 and p29 leading to prolonged and potentially pathological activation . Researchers should be aware that the stability of p35 increases as CDK5 kinase activity decreases, likely resulting from reduced phosphorylation of p35 at Thr138 by CDK5 itself, creating a feedback regulatory mechanism . Phosphorylation of CDK5 at Tyr15 or Ser159 may also modulate activity, although Tyr15 phosphorylation appears to occur primarily on inactive monomeric CDK5 . Light exposure has recently been identified as a regulatory factor for CDK5 activity in the context of circadian rhythm regulation, with CDK5 playing a role in the nuclear import of Period 2 (PER2) . In neurodegenerative conditions like Alzheimer's disease and ALS, aberrant CDK5 hyperactivity has been observed, creating altered experimental parameters that must be accounted for when studying these conditions . When comparing CDK5 activity across different tissues or experimental conditions, researchers should normalize activity to CDK5 protein levels and consider using multiple approaches (activity assays, phospho-specific antibodies, and assessment of downstream substrate phosphorylation) to obtain a comprehensive picture of CDK5 function.

How is CDK5 involved in circadian rhythm regulation and what techniques can researchers use to study this connection?

CDK5 plays a critical role in circadian rhythm regulation through its interaction with key clock components, particularly by facilitating the nuclear import of Period 2 (PER2), an essential step in maintaining circadian oscillations . Researchers investigating this relationship should employ a combination of molecular, cellular, and behavioral approaches. At the molecular level, immunoprecipitation with CDK5 antibodies followed by Western blotting for PER2 can detect physical interactions between these proteins, while in vitro kinase assays can determine if CDK5 directly phosphorylates PER2 or other clock components . Immunofluorescence techniques using both CDK5 and PER2 antibodies can visualize their co-localization and nuclear translocation in the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Studies have demonstrated that silencing Cdk5 using shRNA leads to lack of PER2 expression in the SCN at specific zeitgeber times (ZT), resembling the situation observed in Per2 mutant mice, suggesting CDK5's importance for PER2 stability or expression . At the behavioral level, researchers can use wheel-running activity monitoring in CDK5-modified mice to assess circadian parameters such as period length, phase shifts, and entrainment to light. Recent findings indicate that CDK5 activity is modulated by light and regulates phase shifts of the circadian clock, providing new research avenues for understanding the molecular mechanisms of circadian rhythm disorders .

What is known about CDK5's role in neurodegeneration and how can researchers investigate this using CDK5 antibodies?

CDK5's involvement in neurodegeneration is primarily associated with its dysregulation through formation of the CDK5-p25 complex, which exhibits prolonged activation and altered substrate specificity compared to the normal CDK5-p35 complex. Researchers investigating this aspect should employ antibodies that can specifically distinguish between p35 and its calpain-cleaved product p25, alongside CDK5 antibodies, to monitor the formation of potentially pathological complexes . Immunohistochemistry using CDK5 and p25 antibodies can localize these complexes in brain tissues from patients with neurodegenerative diseases or in animal models, with accumulation of p25 having been documented in Alzheimer's disease and amyotrophic lateral sclerosis (ALS) . To understand the functional consequences of aberrant CDK5 activation, researchers can use phospho-specific antibodies against known CDK5 substrates involved in neurodegeneration, such as tau, neurofilaments, or amyloid precursor protein. Cell culture models exposed to neurotoxic insults that activate calpain and generate p25 can be valuable for studying the mechanisms of CDK5-mediated neurotoxicity and for screening potential therapeutic compounds. Research studies examining CDK5's role in neurodegeneration should include time-course analyses, as the conversion from normal to pathological CDK5 activity is dynamic and potentially reversible in early disease stages .

How can researchers use CDK5 antibodies to study its interactions with other signaling pathways?

Studying CDK5's interactions with other signaling pathways requires sophisticated approaches combining CDK5 antibodies with detection methods for pathway components and activity markers. Co-immunoprecipitation using CDK5 antibodies followed by Western blotting for suspected interaction partners can identify physical associations between CDK5 and components of other pathways. Recent research has revealed connections between CDK5 and the PKA signaling pathway, where CDK5 regulates PKA activity via DARPP32, highlighting the importance of investigating these interconnected networks . Proximity ligation assays (PLA) offer a powerful alternative for visualizing protein-protein interactions in situ, requiring specific antibodies for both CDK5 and its potential interaction partners. For functional studies, researchers can combine CDK5 manipulation (overexpression, knockdown, or inhibition) with activity assays for other pathways to establish cause-effect relationships. Phosphoproteomic approaches using CDK5 antibodies for immunoprecipitation followed by mass spectrometry can identify novel substrates and pathway connections on a larger scale. When investigating CDK5's regulation of transcription factors and gene expression, chromatin immunoprecipitation (ChIP) assays can be performed following CDK5 manipulation to determine how CDK5 activity affects transcription factor binding and chromatin modifications. Research has shown that FoxO1 depletion leads to upregulation of CDK5 and downregulation of the ARF tumor suppressor, indicating complex regulatory relationships that require careful experimental design to untangle .

What are common issues when using CDK5 antibodies and how can they be resolved?

Researchers commonly encounter several issues when working with CDK5 antibodies that require systematic troubleshooting approaches. Non-specific binding is a frequent problem, particularly in Western blotting and immunohistochemistry, which can be addressed by optimizing antibody dilutions (testing a range from 1:500 to 1:5000 for Western blotting and 1:20 to 1:200 for IHC/IF), increasing blocking stringency, or trying different blocking agents such as switching between BSA and non-fat dry milk . Weak or absent signals may result from insufficient antigen retrieval in fixed tissues, requiring optimization of retrieval methods such as heat-induced epitope retrieval or enzymatic retrieval depending on the specific antibody and fixation protocol. Batch-to-batch variability can be problematic, especially with less characterized antibodies, necessitating careful validation of each new lot against positive controls with known CDK5 expression. When using CDK5 antibodies for immunoprecipitation followed by kinase assays, inefficient pull-down may reduce detected activity; this can be improved by using magnetic Dynabeads instead of traditional Protein A/G agarose, optimizing incubation conditions, or testing alternative CDK5 antibodies specifically validated for immunoprecipitation . Cross-reactivity with other CDK family members can confound results, particularly in complex samples, which can be addressed by using antibodies specifically validated to detect endogenous levels of total CDK5 but not CDK1-4 proteins, and by including appropriate controls such as CDK5-depleted samples .

What controls should be included when using CDK5 antibodies for different applications?

Proper controls are essential for interpreting results obtained with CDK5 antibodies across different applications. For Western blotting, researchers should include a positive control (tissue or cell line known to express CDK5, such as neuronal cells), a negative control (CDK5 knockout or knockdown samples), and a molecular weight marker to confirm the expected 30 kDa band for CDK5 . When performing immunoprecipitation, parallel samples using isotype-matched control antibodies (such as AffiniPure Goat Anti-Rabbit IgG or AffiniPure Goat Anti-Mouse IgG, depending on the host species of the CDK5 antibody) should be processed identically to establish background levels . For immunohistochemistry and immunofluorescence, peptide competition controls (pre-incubating the antibody with excess immunizing peptide) can verify specificity, while tissues from CDK5-silenced animals provide definitive negative controls, as demonstrated in studies using shCdk5 in the suprachiasmatic nucleus . When measuring CDK5 kinase activity, essential controls include reactions without substrate, without ATP, and immunoprecipitations using non-specific IgG instead of CDK5 antibody . For studies investigating CDK5's role in specific cellular processes, comparing wild-type conditions with genetic models is invaluable, as illustrated by the comparison between scrambled shRNA, shCdk5, and Per2 Brdm1 mice in circadian rhythm research . Positive controls for antibody functionality, such as including samples with known post-translational modifications when using phospho-specific CDK5 antibodies, help confirm that absence of signal truly represents absence of the modification rather than antibody failure.

What are the best practices for storing and handling CDK5 antibodies to maintain their performance?

Maintaining optimal performance of CDK5 antibodies requires careful attention to storage and handling conditions throughout their lifecycle. Most CDK5 antibodies should be stored according to manufacturer recommendations, typically at -20°C for long-term storage, with aliquoting recommended to avoid repeated freeze-thaw cycles that can degrade antibody quality and lead to reduced specificity and sensitivity . For working solutions, storage at 4°C with appropriate preservatives (such as sodium azide at 0.02%) can maintain stability for several weeks, although regular validation of activity is advisable for critical experiments. Proper handling during experiments includes maintaining cold chain when possible, using clean pipette tips to prevent contamination, and centrifuging antibody solutions before use to remove any aggregates that might cause background issues. Diluted antibody solutions should be prepared fresh in high-quality, filtered buffers, particularly for sensitive applications like immunohistochemistry and immunofluorescence. When working with conjugated CDK5 antibodies (HRP, FITC, or biotin conjugates), protection from light is essential to prevent photobleaching of fluorophores or degradation of enzymes . Researchers should maintain detailed records of antibody performance across different lots and applications, enabling troubleshooting of unexpected results and ensuring experimental reproducibility. For applications requiring exceptional consistency, such as quantitative Western blotting or flow cytometry, validation of each new antibody lot against a reference standard is recommended before use in critical experiments.

How are CDK5 antibodies being used in cancer research and what are the key considerations?

CDK5 antibodies are increasingly used in cancer research as mounting evidence suggests CDK5's involvement in various malignancies through effects on cell proliferation, migration, and DNA damage responses. Researchers studying CDK5 in cancer contexts should consider using both total CDK5 antibodies and phospho-specific antibodies (particularly for Tyr15) to comprehensively assess expression and activation states across tumor samples and cell lines . Immunohistochemical analysis of tumor tissue microarrays with CDK5 antibodies can reveal correlations between CDK5 expression/activation and clinical parameters such as tumor grade, metastatic potential, and patient survival. When investigating mechanistic aspects, co-immunostaining with CDK5 antibodies and markers for proliferation, apoptosis, or DNA damage can provide insights into how CDK5 influences cancer cell behavior. The observation that FoxO1 depletion leads to upregulation of CDK5 and downregulation of the ARF tumor suppressor highlights the complex regulatory relationships that may be relevant in cancer contexts . For translational research, combining CDK5 antibodies with phospho-specific antibodies against cancer-relevant CDK5 substrates can help evaluate the efficacy of CDK5-targeted therapies. Researchers should be mindful of potential differences in CDK5 regulation between normal and cancer cells, as well as between different cancer types, necessitating careful validation of antibodies and experimental approaches for each specific cancer model.

What novel techniques are being developed for studying CDK5 localization and dynamics?

Advanced imaging techniques are revolutionizing the study of CDK5 localization and dynamics, offering unprecedented insights into its spatial and temporal regulation. Super-resolution microscopy approaches such as STORM, PALM, and SIM can be applied with highly specific CDK5 antibodies to visualize CDK5 distribution at nanoscale resolution, revealing previously undetectable details about its subcellular localization and protein-protein interactions. Live-cell imaging using fluorescently tagged CDK5 complemented with antibody-based validation in fixed cells allows researchers to track CDK5 movements in real-time, particularly important for understanding processes like nuclear translocation during circadian rhythm regulation . For studying CDK5 in specific subcellular compartments, researchers are developing fractionation protocols combined with sensitive detection methods using CDK5 antibodies, enabling quantification of CDK5 in nuclear, cytoplasmic, membrane, and synaptic compartments. Proximity labeling techniques such as BioID or APEX, followed by detection with CDK5 antibodies, can map the protein interaction neighborhood of CDK5 in different cellular contexts. Researchers studying CDK5's role in the circadian clock have employed coronal sections of the suprachiasmatic nucleus stained with DAPI, GFP (to identify virus-infected cells), and CDK5 antibodies to visualize CDK5 expression patterns and confirm successful silencing in genetic models . These advanced approaches, when combined with traditional biochemical techniques, provide a more comprehensive understanding of CDK5's dynamic functions across different physiological and pathological states.

How can researchers effectively screen for and validate new CDK5 inhibitors using CDK5 antibodies?

Developing effective CDK5 inhibitors requires robust screening and validation protocols that heavily utilize CDK5 antibodies at multiple stages. Initial screening assays typically employ in vitro kinase reactions with immunoprecipitated CDK5 (using specific CDK5 antibodies) and appropriate substrates, allowing measurement of inhibitor potency against purified CDK5 complexes . Following identification of promising compounds, cellular validation using Western blotting with phospho-specific antibodies against known CDK5 substrates can confirm target engagement and pathway inhibition in intact cells. For assessing selectivity, parallel kinase assays with related CDKs followed by Western blotting with antibodies specific to each CDK can determine inhibitor specificity across the CDK family. Researchers should leverage the observation that specific CDK5 antibodies can detect endogenous levels of total CDK5 protein but not recombinant CDK1-4 proteins, providing a foundation for distinguishing CDK5-specific effects . Competition binding assays between inhibitors and CDK5 antibodies can provide structural insights into inhibitor binding sites if the epitope recognized by the antibody is known. For in vivo validation, immunohistochemistry with CDK5 and phospho-substrate antibodies in tissues from inhibitor-treated animals can demonstrate target engagement in physiologically relevant contexts. Advanced approaches may include chemoproteomics with activity-based probes coupled with CDK5 antibodies for detection, allowing proteome-wide profiling of inhibitor selectivity and potency.

What are the typical applications and recommended dilutions for different CDK5 antibodies?

Different CDK5 antibodies are optimized for specific applications, with recommended dilutions varying based on the antibody's format, concentration, and the specific application. Based on the available research data, the following table summarizes typical applications and recommended dilutions for various CDK5 antibodies:

For CDK5 antibodies with specific conjugates, additional considerations apply:

When working with phospho-specific CDK5 antibodies (such as those targeting pTyr15), researchers typically need to use more concentrated antibody solutions (lower dilutions) and should consider using phosphatase inhibitors in all buffers to preserve the phosphorylation state .

What are the key characteristics of commonly used CDK5 monoclonal antibodies?

Several CDK5 monoclonal antibodies are commonly used in research, each with distinct characteristics that make them suitable for specific applications. The following table summarizes key properties of well-characterized CDK5 monoclonal antibodies based on the search results:

These antibodies offer researchers various options depending on their specific experimental needs, with some optimized for particular applications such as immunoprecipitation for kinase assays, while others provide broader utility across multiple techniques. The specificity of antibodies like Cell Signaling #2506, which detects endogenous CDK5 but not related CDKs, is particularly valuable for studies requiring clear discrimination between these structurally similar kinases .

What model systems and databases are useful for CDK5 research?

Researchers studying CDK5 have access to various model systems and databases that facilitate comprehensive investigation of its functions and interactions. The following table outlines key resources for CDK5 research:

These resources collectively provide researchers with standardized systems for investigating CDK5 biology, from genetic manipulation to biochemical characterization. The availability of well-characterized antibodies against CDK5 and its regulators, combined with established protocols and database resources, enables comprehensive analysis of CDK5's diverse functions across multiple experimental contexts.