CDC37 Antibody

What is CDC37 and what cellular functions does it perform?

CDC37 (Cell Division Cycle 37) is a molecular chaperone with specific functions in cell signal transduction. It plays a crucial role in cell cycle regulation by facilitating the formation of cyclin-dependent kinase 4 (Cdk4) and cyclin D1 complex, which is essential for G1 to S phase transition . CDC37 forms a stable complex with heat shock protein 90 (Hsp90), important for the stability and function of various client proteins involved in cell cycle regulation . Proper functioning of CDC37 and its associated protein interactions are critical for maintaining cellular homeostasis and preventing uncontrolled cell proliferation, which can lead to cancer .

What types of CDC37 antibodies are available for research applications?

Researchers have access to several types of CDC37 antibodies:

These antibodies are offered in multiple configurations to support diverse experimental needs across western blotting, immunoprecipitation, immunofluorescence, immunohistochemistry, and ELISA applications .

How should researchers optimize CDC37 antibody use in western blotting protocols?

For optimal western blotting results with CDC37 antibodies, researchers should implement the following methodological considerations:

• Sample preparation: Use fresh samples with complete protease inhibitor cocktails to prevent degradation of CDC37 protein.

• Loading controls: Include appropriate loading controls (β-actin, GAPDH) to normalize CDC37 expression levels.

• Blocking: Use 5% non-fat dry milk or BSA in TBS-T for 1 hour at room temperature to minimize background.

• Primary antibody dilution: For monoclonal antibodies like CDC37 (E-4), start with 1:200-1:1000 dilution and optimize based on signal strength .

• Incubation conditions: Overnight incubation at 4°C generally yields better results than shorter incubations at room temperature.

• Validation controls: Include positive control lysates from cells known to express CDC37 and negative controls where CDC37 has been depleted through siRNA.

• Detection method selection: For highest sensitivity, consider using HRP-conjugated antibodies with enhanced chemiluminescence detection .

When troubleshooting weak signals, increasing antibody concentration or extending incubation time often improves results, while high background may require more stringent washing or adjusting blocking conditions.

What are the proper sample preparation techniques for immunoprecipitation of CDC37-containing complexes?

Successful immunoprecipitation of CDC37 complexes requires preserving native protein interactions:

• Cell lysis buffer selection: Use non-denaturing buffers containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40 or Triton X-100, with protease and phosphatase inhibitors.

• Lysis conditions: Maintain samples at 4°C throughout processing to preserve protein-protein interactions, particularly CDC37-HSP90 complexes.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody selection: For CDC37 immunoprecipitation, agarose-conjugated antibodies like CDC37 Antibody (E-4) AC provide consistent results .

• Cross-linking consideration: For weak or transient interactions, consider using chemical cross-linkers prior to lysis.

• Elution strategy: Mild elution conditions help maintain complex integrity for downstream analysis.

• Controls: Always include an isotype control antibody (e.g., mouse IgG2b for CDC37 (E-4) antibody) to identify non-specific binding .

These methodological refinements are essential for accurately studying CDC37's interactions with client proteins and HSP90.

How can researchers distinguish between intracellular and cell surface CDC37?

Discriminating between intracellular and cell surface CDC37 pools requires specialized approaches:

• Cell surface biotinylation: Use cell-impermeable biotinylation reagents to selectively label surface proteins, followed by streptavidin pull-down and CDC37 immunoblotting.

• Cell-impermeable antibodies: Employ cell-impermeable antibodies that can only access surface CDC37, as demonstrated in studies with breast cancer cell lines MDA-MB-453 and MDA-MB-231 .

• Immunofluorescence without permeabilization: Perform initial staining without detergents to detect only surface CDC37, followed by permeabilization and re-staining to visualize total CDC37.

• Flow cytometry with differential permeabilization: Compare CDC37 staining in permeabilized versus non-permeabilized cells to quantify surface versus total CDC37.

• Confocal microscopy with Z-stack analysis: Utilize optical sectioning to distinguish membrane-localized from cytoplasmic CDC37.

Research has established that CDC37 exists both intracellularly and on the cell surface of cancer cells, where surface CDC37 participates in cancer cell motility processes and interacts with HSP90 and kinase receptors including HER2 and EGFR .

What experimental approaches reveal CDC37's role in non-kinase protein stabilization?

While CDC37 traditionally functions as a kinase-specific co-chaperone, recent research demonstrates its involvement with non-kinase clients:

• Viral protein stability assays: Studies with rabies virus (RABV) have shown that CDC37 chaperones the non-kinase phosphoprotein (P) during infection .

• Inhibition studies: Activity inhibition and knockdown of CDC37 and HSP90 increased instability of viral P protein, confirming their chaperoning function .

• Overexpression experiments: CDC37 and HSP90 overexpression maintained P protein stability without increasing infectious virus yield .

• Co-immunoprecipitation: This technique demonstrated that CDC37, whether phosphorylated or unphosphorylated on Ser13, aids in loading client proteins onto HSP90 machinery .

• Allosteric regulation analysis: Research suggests that CDC37-HSP90 interaction influences conformational switches in HSP90, affecting chaperoning activity .

These findings highlight a novel mechanism where CDC37/HSP90 chaperones non-kinase targets, with significant implications for understanding both chaperone biology and potential antiviral therapeutic strategies .

How does CDC37 contribute to cancer cell invasion and metastatic processes?

CDC37's role in cancer progression extends beyond its intracellular functions:

• Cell surface localization studies have revealed that CDC37 is present on the surface of breast cancer cells, where it participates in cancer cell motility processes .

• Protein complex analysis demonstrates that surface CDC37 interacts with HSP90 and kinase receptors (HER2, EGFR) on the cell surface, acting as a co-factor in HSP90's extracellular chaperoning activities .

• Functional inhibition experiments using cell-impermeable antibodies against surface HSP90 (mAb 4C5) showed disruption of both CDC37/HSP90 complex and CDC37/ErbB receptor complexes .

• Invasion assays confirmed that surface CDC37, in concert with HSP90, plays an essential role during cancer cell invasion processes .

These findings support a model where surface CDC37 facilitates cancer cell invasion through extracellular chaperoning activities, providing potential new therapeutic targets for metastasis inhibition .

What are the most common sources of false positives/negatives when using CDC37 antibodies?

Researchers should be aware of several potential artifacts when using CDC37 antibodies:

• False positives:

  • Cross-reactivity with structurally similar proteins

  • Non-specific binding in high-expression systems

  • Signal from secondary antibody binding to endogenous immunoglobulins

  • Degradation products appearing as multiple bands

• False negatives:

  • Epitope masking due to protein-protein interactions

  • Post-translational modifications affecting antibody recognition

  • Inadequate protein extraction from samples

  • Suboptimal transfer conditions for high molecular weight complexes

For validation, researchers should:

• Use multiple antibodies targeting different CDC37 epitopes

• Include positive and negative controls (knockout/knockdown)

• Verify results with complementary techniques (mass spectrometry)

• Consider species-specific reactivity (human CDC37 shares 95.7% and 95.1% amino acid sequence identity with mouse and rat CDC37)

What controls are essential when studying CDC37-HSP90 interactions?

Rigorous experimental design for CDC37-HSP90 interaction studies requires multiple controls:

These controls help distinguish true interactions from experimental artifacts and provide critical context for interpreting results in the CDC37-HSP90 chaperone system .

How is our understanding of CDC37 evolving beyond its classical co-chaperone role?

Recent research is expanding CDC37's known functions beyond its classical role:

• Non-kinase client chaperoning: CDC37's ability to stabilize non-kinase proteins like rabies virus phosphoprotein reveals broader substrate recognition than previously thought .

• Extracellular functions: The discovery of cell surface CDC37 on cancer cells introduces entirely new functional domains, particularly in cancer cell motility and invasion .

• Allosteric regulation: CDC37's effect on HSP90 conformational switches suggests more complex regulatory mechanisms than simple client protein loading .

• Phosphorylation-independent functions: The finding that both phosphorylated and unphosphorylated CDC37 can aid in client protein loading challenges previous models of CDC37 regulation .

• Cancer therapeutic targeting: Surface CDC37's role in cancer cell invasion processes makes it a potential therapeutic target, with preliminary evidence supporting antibody-based approaches .

These discoveries suggest CDC37 has more diverse cellular functions than previously recognized, opening new research directions in both normal cellular processes and disease states.

What technological advancements are improving CDC37 antibody-based research?

Several cutting-edge technologies are enhancing CDC37 antibody applications:

• Super-resolution microscopy allows visualization of CDC37-containing complexes at nanometer resolution, revealing previously undetectable spatial relationships between CDC37, HSP90, and client proteins.

• Multicolor flow cytometry with surface-specific antibodies enables quantitative analysis of surface versus intracellular CDC37 pools across different cell populations.

• Proximity ligation assays provide sensitive detection of CDC37-protein interactions in situ with single-molecule resolution.

• Cell-impermeable antibody conjugates with novel fluorophores, nanoparticles, or therapeutic agents expand the utility of surface CDC37 detection for both research and potential clinical applications .

• Single-cell proteomics combined with CDC37 antibodies allows analysis of CDC37 expression and complex formation at the individual cell level, revealing population heterogeneity.

These technological advances are facilitating more precise investigations into CDC37's diverse functions across different cellular compartments and disease states.