Phospho-CDC37 (S13) Recombinant Monoclonal Antibody

What is the biological significance of CDC37 phosphorylation at S13?

CDC37 phosphorylation at serine 13 plays a crucial role in promoting the formation of stable HSP90-CDC37-kinase ternary complexes, which are essential for proper kinase folding and activation. Importantly, S13 phosphorylation does not affect the boundaries of CDC37's predicted secondary structure elements or its ability to form binary CDC37-kinase complexes. Instead, this post-translational modification appears to specifically stabilize the interaction with HSP90, functioning at a later stage in the chaperone cycle after initial kinase recognition . The phosphorylation event does not alter the mode of kinase recognition or binding affinity between CDC37 and kinases, but rather regulates the stability of the ternary complex formation .

What detection methods are compatible with the Phospho-CDC37 (S13) antibody?

The Phospho-CDC37 (S13) Recombinant Monoclonal Antibody has been validated for dot blots and Western blot (WB) analyses . For Western blot applications, the antibody has been tested at a 1/10000 dilution and successfully detects the phosphorylated form of CDC37 at the predicted band size of 44 kDa. Researchers should note that the antibody has been specifically tested with human, mouse, and rat samples, showing consistent results across these species . When conducting Western blot experiments, the antibody can be paired with appropriate secondary antibodies such as Goat Anti-Rabbit IgG H&L (HRP) at a 1/20000 dilution .

How is specificity for phosphorylated S13 validated in experimental settings?

Specificity validation for the Phospho-CDC37 (S13) antibody typically involves phosphatase treatment controls. In Western blot experiments, researchers compare samples with and without phosphatase treatment to confirm specificity for the phosphorylated form of the protein. As demonstrated in validation data, the antibody shows strong reactivity with phosphorylated CDC37 in untreated lysates, while the signal disappears or significantly decreases in phosphatase-treated samples . Additional validation involves using Calyculin A, a phosphatase inhibitor that enhances S13 phosphorylation levels, resulting in stronger signal detection in treated versus untreated samples .

How does S13 phosphorylation impact the structural dynamics of CDC37 in complex formation?

S13 phosphorylation of CDC37 does not alter the protein's mode of kinase recognition or binding affinity. NMR spectroscopy analysis reveals that phosphorylated and non-phosphorylated forms of CDC37 produce identical chemical shift perturbation patterns when interacting with client kinases, such as bRaf . Both forms exhibit the same thermodynamic signatures of binding and identical affinities for client kinases. The current model suggests that S13 phosphorylation primarily functions at later stages of the chaperone cycle, specifically in stabilizing ternary complexes with HSP90 rather than affecting initial kinase recognition . The phosphorylation event appears to act as a regulatory switch that promotes progression through the chaperone cycle after the initial CDC37-kinase binding has occurred.

What are the key experimental considerations when comparing phosphorylated versus non-phosphorylated CDC37 in kinase recognition studies?

When designing experiments to compare phosphorylated versus non-phosphorylated CDC37 in kinase recognition studies, researchers should consider the following methodological approaches:

• Phosphomimetic mutations: Use S13D or S13E mutations to mimic constitutive phosphorylation and S13A to prevent phosphorylation

• Controlled phosphorylation: Employ CK2 kinase for in vitro phosphorylation of recombinant CDC37

• Biochemical validation: Confirm phosphorylation status using the phospho-specific antibody alongside mass spectrometry

• Binding studies: Implement multiple techniques (ITC, SPR, NMR) to measure binding constants

• Functional assays: Design experiments that specifically distinguish between binary (CDC37-kinase) and ternary (HSP90-CDC37-kinase) complex formation

Analysis should account for the fact that S13 phosphorylation impacts ternary complex stability without altering binary CDC37-kinase complex formation . Additionally, the S13A mutation affects client interaction and maturation primarily at later stages involving HSP90, with minimal impact on binary complex formation .

How can the Phospho-CDC37 (S13) antibody be used to investigate the role of CDC37 in oncogenic kinase stabilization?

The Phospho-CDC37 (S13) antibody provides a valuable tool for investigating CDC37's involvement in oncogenic kinase stabilization through several methodological approaches:

• Comparative analysis of phosphorylation levels: Quantify S13 phosphorylation across normal versus cancer cell lines or tissues to establish correlation with oncogenic transformation

• Kinase-specific interactions: Perform co-immunoprecipitation experiments using the phospho-specific antibody to isolate and identify associated kinases in various cancer models

• Oncogenic versus wild-type kinase comparisons: As demonstrated with bRaf versus bRaf V600E, CDC37 shows stronger interaction with oncogenic variants ; the phospho-specific antibody can help quantify these differential interactions

• Pharmacological intervention studies: Monitor changes in S13 phosphorylation following treatment with HSP90 inhibitors, kinase inhibitors, or phosphatase modulators

Research data indicates that oncogenic kinases such as bRaf V600E show significantly stronger interaction with CDC37 compared to their wild-type counterparts . This suggests that phosphorylated CDC37 may play a more critical role in stabilizing oncogenic kinases, making the Phospho-CDC37 (S13) antibody particularly valuable for cancer research applications.

What are the optimal sample preparation protocols for detecting Phospho-CDC37 (S13) in different experimental systems?

For optimal detection of Phospho-CDC37 (S13) in various experimental systems, researchers should implement the following sample preparation protocols:

Cell Culture Samples:

• Treat cells with phosphatase inhibitors (50nM Calyculin A for 3 hours) to preserve phosphorylation status

• Harvest cells in buffer containing phosphatase inhibitor cocktails

• Lyse cells using buffers containing detergents suitable for membrane disruption (RIPA or NP-40)

• Centrifuge lysates at 14,000g for 15 minutes at 4°C to remove cellular debris

• Quantify protein concentration using Bradford or BCA assays

• Load 15-20 μg of total protein per lane for Western blot analysis

Tissue Samples:

• Snap-freeze tissues immediately after collection

• Homogenize in cold lysis buffer containing phosphatase inhibitors

• Clarify lysates by centrifugation

• Process as with cell culture samples

For validation purposes, researchers should prepare parallel samples treated with phosphatase to confirm antibody specificity for the phosphorylated form of CDC37 .

What experimental controls are essential when using the Phospho-CDC37 (S13) antibody in client kinase interaction studies?

When utilizing the Phospho-CDC37 (S13) antibody in client kinase interaction studies, the following controls are essential for data validation:

Using these controls helps ensure reliable interpretation of results by distinguishing specific phospho-CDC37 (S13) signals from potential artifacts or non-specific binding.

How can Phospho-CDC37 (S13) antibody be used in multiplexed detection systems with other chaperone complex components?

For multiplexed detection of Phospho-CDC37 (S13) alongside other chaperone complex components, researchers can employ several methodological approaches:

• Sequential immunoblotting: Strip and reprobe membranes with antibodies against HSP90, client kinases, and other co-chaperones, ensuring appropriate molecular weight separation

• Fluorescent multiplex Western blotting: Utilize primary antibodies from different species and corresponding fluorescently-labeled secondary antibodies with distinct excitation/emission profiles

• Co-immunoprecipitation followed by immunoblotting:

  • Immunoprecipitate with Phospho-CDC37 (S13) antibody

  • Probe precipitates for HSP90, client kinases, and other components

  • Compare with reciprocal immunoprecipitations using antibodies against complex components

• Multiplex immunofluorescence microscopy:

  • Use differently labeled secondary antibodies against primaries for Phospho-CDC37 (S13), HSP90, and client kinases

  • Analyze co-localization patterns using confocal microscopy

  • Quantify colocalization coefficients to assess complex formation

• Proximity ligation assay (PLA):

  • Combine Phospho-CDC37 (S13) antibody with antibodies against HSP90 or kinases

  • Generate fluorescent signals only when proteins are in close proximity (<40nm)

  • Quantify interaction events in situ

These multiplexed approaches allow researchers to simultaneously examine the phosphorylation status of CDC37 and its association with other components of the chaperone machinery in various experimental contexts.

How should researchers interpret changes in CDC37 S13 phosphorylation levels in response to different cellular stresses?

Interpreting changes in CDC37 S13 phosphorylation in response to cellular stresses requires consideration of multiple factors. CDC37 phosphorylation at S13 promotes the formation of stable HSP90-CDC37-kinase ternary complexes essential for kinase maturation and function . Therefore, changes in phosphorylation levels may indicate adaptive responses in the cell's protein quality control system.

When analyzing phosphorylation changes, researchers should:

Increases in S13 phosphorylation often indicate enhanced demand for kinase stabilization, while decreased phosphorylation may suggest reduced chaperone capacity or altered regulatory mechanisms in the stress response.

What are common technical challenges when detecting Phospho-CDC37 (S13) and their solutions?

Researchers commonly encounter several technical challenges when working with Phospho-CDC37 (S13) antibody. The following table outlines these challenges and provides methodological solutions:

When troubleshooting, researchers should remember that the Phospho-CDC37 (S13) antibody has been validated specifically for dot blots and Western blot applications at defined concentrations (1/10000 dilution) .

How does Phospho-CDC37 (S13) status correlate with the stability and activity of client kinases across different experimental models?

The correlation between Phospho-CDC37 (S13) status and client kinase stability/activity varies across experimental models, reflecting the complex regulation of the HSP90-CDC37-kinase chaperone system. Research findings indicate:

• Cancer models: Enhanced S13 phosphorylation often correlates with increased stability of oncogenic kinases like bRaf V600E, which shows significantly stronger interaction with CDC37 compared to wild-type bRaf . This suggests that phosphorylated CDC37 may preferentially stabilize oncogenic kinase variants.

• Cell type specificity: Different cell types show variable dependency on CDC37 phosphorylation for kinase stability, likely reflecting tissue-specific chaperone networks.

• Kinase-specific effects: The impact of S13 phosphorylation appears to be more pronounced for certain kinases. While S13 phosphorylation does not directly affect the mode of kinase recognition or binding affinity of CDC37 to kinases like bRaf , it enhances stability of ternary complexes involving HSP90.

• Temporal dynamics: The relationship between phosphorylation and kinase activity shows time-dependent patterns during cellular processes like differentiation and stress response.

• Pharmacological interventions: HSP90 inhibitors can disrupt the stability provided by phosphorylated CDC37, leading to degradation of client kinases, suggesting that targeting this phosphorylation could be a therapeutic strategy.

When designing experiments to investigate these correlations, researchers should employ multiple approaches, including phosphorylation-site mutants (S13A, S13D), pharmacological modulators of CK2 activity, and client kinase activity/stability assays .

How can researchers utilize the Phospho-CDC37 (S13) antibody to investigate the conformational changes in CDC37 upon phosphorylation?

Investigating conformational changes in CDC37 upon S13 phosphorylation requires sophisticated biophysical approaches combined with antibody-based detection. Researchers can employ the following methodological strategies:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Compare exchange patterns between phosphorylated and non-phosphorylated CDC37

  • Use the Phospho-CDC37 (S13) antibody to validate phosphorylation status of samples

  • Correlate regions of altered solvent accessibility with functional domains

• Limited proteolysis coupled with Western blotting:

  • Subject phosphorylated and non-phosphorylated CDC37 to controlled proteolytic digestion

  • Analyze fragment patterns using the Phospho-CDC37 (S13) antibody and total CDC37 antibodies

  • Identify regions with altered protease sensitivity indicating conformational differences

• Cross-linking mass spectrometry:

  • Apply chemical cross-linkers to capture the three-dimensional structure

  • Identify crosslinked peptides by mass spectrometry

  • Compare crosslinking patterns between phosphorylated and non-phosphorylated forms

• Single-molecule FRET combined with immunovalidation:

  • Engineer CDC37 with fluorophore pairs at strategic positions

  • Measure FRET efficiency changes upon phosphorylation

  • Validate phosphorylation status using the Phospho-CDC37 (S13) antibody

Current evidence indicates that S13 phosphorylation does not significantly alter the predicted secondary structure elements of N-Cdc37 , suggesting that its effects may involve more subtle conformational changes or alterations in protein-protein interaction surfaces that facilitate stable ternary complex formation with HSP90 and client kinases.

What approaches can be used to investigate the differential effects of S13 phosphorylation on various client kinase interactions?

To investigate how S13 phosphorylation differentially affects interactions with various client kinases, researchers can implement the following methodological approaches:

• Comparative binding assays:

  • Perform quantitative pull-down experiments with phosphorylated versus non-phosphorylated CDC37

  • Compare binding affinities across a panel of client and non-client kinases

  • Use the Phospho-CDC37 (S13) antibody to confirm phosphorylation status

• Kinase specificity profiling:

  • Develop protein microarrays with diverse kinases

  • Probe with phosphorylated versus non-phosphorylated CDC37

  • Quantify binding differences using detection systems including the Phospho-CDC37 (S13) antibody

• Structural characterization of complexes:

  • Use cryo-EM to visualize CDC37-kinase complexes with and without S13 phosphorylation

  • Perform hydrogen-deuterium exchange mass spectrometry on complexes with different kinases

  • Identify kinase-specific interaction interfaces affected by phosphorylation

• Client kinase stability assays:

  • Express CDC37 wild-type, S13A, and S13D/E mutants in cells

  • Measure half-lives of different client kinases under each condition

  • Correlate with phosphorylation status using the Phospho-CDC37 (S13) antibody

Research indicates that while S13 phosphorylation does not alter binary CDC37-kinase complex formation, it significantly impacts the formation of stable ternary complexes with HSP90 . The effect appears to be more pronounced with certain kinases, particularly oncogenic variants like bRaf V600E, which shows stronger interaction with CDC37 compared to wild-type bRaf .

How can multi-omics approaches incorporating Phospho-CDC37 (S13) detection advance our understanding of chaperone networks in disease models?

Multi-omics approaches integrating Phospho-CDC37 (S13) detection can significantly enhance our understanding of chaperone networks in disease models through systematic analysis of multiple biological layers:

• Phosphoproteomics and Interactomics integration:

  • Perform phospho-enrichment coupled with mass spectrometry across disease models

  • Use the Phospho-CDC37 (S13) antibody for validation and quantification

  • Correlate CDC37 S13 phosphorylation with global phosphorylation networks

  • Perform parallel interactome analysis using co-immunoprecipitation with the phospho-specific antibody

  • Construct interaction networks specific to phosphorylated versus non-phosphorylated CDC37

• Transcriptomics and Proteomics correlation:

  • Compare transcriptional profiles of cells with normal versus altered CDC37 phosphorylation

  • Correlate with protein abundance changes of chaperone components and client kinases

  • Use the Phospho-CDC37 (S13) antibody to stratify samples based on phosphorylation status

• Functional genomics screens:

  • Perform CRISPR screens to identify genes affecting CDC37 S13 phosphorylation

  • Use the phospho-specific antibody as a readout for screen validation

  • Connect genetic dependencies with CDC37 phosphorylation status in disease models

• Metabolomics integration:

  • Correlate metabolic signatures with CDC37 phosphorylation status

  • Investigate how metabolic states influence chaperone function via CDC37 modification

  • Examine feedback loops between kinase signaling and metabolism mediated by CDC37

This multi-dimensional analysis can reveal how CDC37 S13 phosphorylation serves as a regulatory node within larger cellular networks, potentially identifying new therapeutic targets for diseases dependent on specific chaperone-client interactions.