CDC37L1 Antibody

What is CDC37L1 and why is it relevant to cancer research?

CDC37L1 (also known as CDC37B or HARC) is a co-chaperone protein that contains 337 amino acids and is structurally similar to CDC37. Unlike its analog CDC37 (which promotes tumor progression), CDC37L1 functions as a tumor suppressor in several cancer types. Research shows that CDC37L1 expression is decreased in high-grade gastric cancer tissues compared to low-grade tissues, and lower expression correlates with poor patient survival rates . CDC37L1 contributes to the regulation of HSP90 function and appears to inhibit cancer cell proliferation and migration through various mechanisms, most notably through CDK6 reduction .

What applications can CDC37L1 antibodies be used for in laboratory research?

CDC37L1 antibodies have been validated for multiple laboratory applications:

For optimal results, these antibodies have been successfully tested on various human samples including HeLa cells, Jurkat cells, MCF-7 cells, U2OS cells, and human kidney and liver tissues . When performing IHC, antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0 is recommended for best results .

How should CDC37L1 antibodies be stored and handled to maintain reactivity?

CDC37L1 antibodies typically come in liquid form in PBS containing 50% glycerol, 0.5% BSA and 0.02% sodium azide at pH 7.3 . For optimal stability:

• Store antibodies at -20°C where they remain stable for approximately one year after shipment

• For smaller antibody quantities (≤20μl), aliquoting is generally unnecessary for -20°C storage

• For larger volumes, consider creating single-use aliquots to avoid repeated freeze-thaw cycles

• Upon receipt of new antibodies, some manufacturers recommend storing at -80°C for longest shelf-life

• Always centrifuge briefly before opening the antibody vial to ensure collection of all liquid

What controls should be included when validating CDC37L1 antibody specificity?

Proper controls are essential for validating CDC37L1 antibody specificity:

• Positive controls: Use cell lines known to express CDC37L1 such as HeLa, Jurkat, MCF-7, or U2OS cells

• Negative controls: Consider primary antibody omission controls and isotype controls

• Knockdown/knockout validation: Implement CDC37L1 siRNA knockdown or CRISPR/Cas9 knockout samples as gold-standard specificity controls

• Molecular weight verification: Confirm detection at the expected molecular weight of approximately 39 kDa

• Tissue expression patterns: Compare results with known CDC37L1 expression patterns (expressed in kidney and liver tissues)

Researchers should validate each new lot of antibody in their specific experimental system before use in critical experiments.

What are the optimal conditions for Western blot detection of CDC37L1?

For optimal Western blot detection of CDC37L1:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for efficient extraction

  • Load 20-40 μg of total protein per lane

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal resolution of the 39 kDa CDC37L1 protein

• Transfer conditions:

  • Semi-dry or wet transfer systems work well

  • Transfer at 100V for 90 minutes or 30V overnight in cold room

• Blocking and antibody incubation:

  • Block membranes with 5% non-fat dry milk or BSA in TBST

  • Dilute primary CDC37L1 antibody between 1:1000-1:5000 based on signal strength required

  • Incubate overnight at 4°C for best results

  • Use appropriate HRP-conjugated secondary antibody at 1:5000-1:10000 dilution

• Signal detection:

  • Enhanced chemiluminescence (ECL) systems are suitable for detection

  • Expected molecular weight is 39 kDa

How should researchers approach CDC37L1 detection in tissue microarrays by IHC?

Based on published research using CDC37L1 antibodies in tissue microarrays :

• Antigen retrieval:

  • Use TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0

  • Heat-induced epitope retrieval methods yield best results

• Antibody dilution and incubation:

  • Optimize dilutions between 1:50-1:500 depending on antibody sensitivity

  • Incubate overnight at 4°C in a humidified chamber

• Scoring system for expression levels:

  • Evaluate both staining intensity (weak, moderate, strong) and percentage of positive cells

  • Use two independent pathologists for scoring to reduce subjective bias

  • Consider automated image analysis systems for quantitative assessment

• Controls and reporting:

  • Include positive control tissues (kidney, liver) on each slide

  • Thoroughly document methodology including antibody source, clone/lot, dilution, incubation time, and scoring system

  • Report both tissue and subcellular localization patterns

How can CDC37L1 antibodies be used to study its role in cancer progression mechanisms?

CDC37L1 antibodies can be utilized in multiple sophisticated approaches to investigate its mechanistic role in cancer:

• Co-immunoprecipitation (Co-IP):

  • Use CDC37L1 antibodies to pull down protein complexes and identify interaction partners

  • This approach revealed CDC37L1's interaction with HSP90 and its client proteins

  • Western blot analysis following Co-IP can detect specific interactions with proteins like CDK6

• Chromatin immunoprecipitation (ChIP):

  • Investigate whether CDC37L1 associates with chromatin-bound complexes

  • May help elucidate its role in transcriptional regulation

• Proximity ligation assay (PLA):

  • Visualize protein-protein interactions in situ

  • Particularly useful for confirming interactions between CDC37L1 and potential partners like HSP90

• Immunofluorescence microscopy:

  • Track subcellular localization changes during cancer progression

  • Combine with other markers to assess co-localization with client proteins

• Proteomic approaches:

  • Use antibody-based enrichment followed by mass spectrometry

  • Can identify novel CDC37L1-associated proteins in different cancer contexts

These approaches have helped establish CDC37L1's role in inhibiting gastric cancer cell proliferation and migration through CDK6 reduction .

What is known about CDC37L1's relationship with HSP90 and how can this be studied?

CDC37L1 functions as a co-chaperone for HSP90, potentially regulating its function . This relationship can be investigated using:

• In vitro binding assays:

  • Pull-down assays using recombinant proteins

  • Surface plasmon resonance to measure binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

• Cellular assays:

  • Co-IP with CDC37L1 antibodies followed by HSP90 detection

  • Manipulation of CDC37L1 levels (overexpression/knockdown) to assess impact on HSP90 client protein stability

  • Assess whether CDC37L1 competes with CDC37 for HSP90 binding

• Functional assays:

  • ATPase activity assays to determine if CDC37L1 affects HSP90's enzymatic function

  • Client protein folding assays in the presence/absence of CDC37L1

  • HSP90 inhibitor sensitivity in cells with varying CDC37L1 expression

Research has shown that CDC37L1 strengthens the binding between HSP90 and certain client proteins like PPIA (peptidylprolyl isomerase A), suggesting it may selectively modulate HSP90's chaperone function toward specific client proteins .

How does CDC37L1 expression influence drug resistance mechanisms in cancer therapy?

CDC37L1 has been implicated in drug resistance mechanisms, particularly regarding sorafenib resistance in hepatocellular carcinoma (HCC):

• Expression correlation with therapy response:

  • High expression of CDC37L1 and its downstream effector PPIA predicts worse prognosis in HCC patients undergoing sorafenib therapy

  • CDC37L1 appears to enhance sorafenib resistance in HCC cells both in vitro and in vivo

• Mechanistic insights:

  • CDC37L1, as a cochaperone, increases expression of PPIA by strengthening the binding between HSP90 and PPIA

  • This protein interaction network appears to contribute to therapy resistance mechanisms

• MicroRNA regulation:

  • CDC37L1 is negatively regulated by miR-15a and miR-20b

  • Exogenous expression of these miRNAs enhances sorafenib sensitivity in HCC cells

  • This suggests a regulatory axis of miR-15a/miR-20b → CDC37L1 → PPIA that influences drug response

• Potential therapeutic targets:

  • Targeting CDC37L1 or its regulatory miRNAs might be a strategy to overcome sorafenib resistance

  • Combined inhibition of HSP90 and modulation of CDC37L1 levels could sensitize resistant tumors

How should researchers address contradictory findings regarding CDC37L1 in different cancer types?

When encountering conflicting data about CDC37L1's role across cancer types:

• Methodological considerations:

  • Different antibodies may have varying specificities and sensitivities

  • Compare antibody clones, dilutions, and detection methods used across studies

  • Evaluate sample preparation techniques and scoring systems in IHC studies

• Biological context differences:

  • CDC37L1 functions may be tissue-specific and context-dependent

  • In gastric cancer, CDC37L1 acts as a tumor suppressor

  • In hepatocellular carcinoma, high CDC37L1 expression correlates with sorafenib resistance

  • Consider the molecular subtype of cancer being studied

• Genetic background influences:

  • Analyze whether genetic alterations in related pathways modify CDC37L1 function

  • Consider oncogenic drivers present in different tumor types (e.g., RAS mutations)

• Technical validation approaches:

  • Use multiple detection methods (IHC, WB, qPCR) to confirm expression findings

  • Employ functional studies (knockdown/overexpression) to validate biological effects

  • Consider spatial heterogeneity in tumors that might affect sampling

• Data integration strategies:

  • Correlate findings with public databases (e.g., TCGA) for broader context

  • Meta-analysis approaches may help resolve contradictions

  • Single-cell analysis can reveal heterogeneity masked in bulk tissue studies

What patterns of CDC37L1 expression have been observed across cancer progression stages?

Research has revealed specific patterns of CDC37L1 expression across cancer progression:

Understanding these patterns has important implications for using CDC37L1 as a prognostic biomarker and potential therapeutic target.

How can CDC37L1 antibodies be utilized in screening for novel therapeutic approaches?

CDC37L1 antibodies enable several innovative screening strategies:

• Antibody-drug conjugate (ADC) development:

  • While not directly related to CDC37L1, similar approaches targeting cell surface proteins like CDCP1 have shown promise in RAS-driven cancers

  • CDC37L1-interacting proteins might be targetable with ADCs if they show cancer-specific expression

• High-throughput compound screening:

  • CDC37L1 antibodies can be used in cell-based assays to screen for compounds that modulate its expression or function

  • Changes in CDC37L1 levels or localization following compound treatment can be detected by immunofluorescence or high-content imaging

• PROTAC (Proteolysis Targeting Chimera) development:

  • CDC37L1 antibodies can help validate degradation of target proteins in PROTAC development

  • Monitor changes in CDC37L1 client proteins following targeted degradation approaches

• Synthetic lethal interactions:

  • CDC37L1 knockdown combined with drug libraries can identify synthetic lethal interactions

  • CRISPR/Cas9 screening approaches have identified CDC37L1 as involved in sorafenib resistance

• Combination therapy evaluation:

  • CDC37L1 antibodies can monitor expression changes during combination treatments

  • Particularly relevant for combinations of HSP90 inhibitors with other targeted therapies

What are the latest techniques for studying CDC37L1 interactions with the cell cycle machinery?

Advanced techniques for investigating CDC37L1's role in cell cycle regulation include:

• Live-cell imaging approaches:

  • Fluorescently tagged CDC37L1 to track localization during cell cycle progression

  • FRET/BRET-based biosensors to detect interactions with cell cycle proteins in real-time

• Mass spectrometry-based interactomics:

  • BioID or APEX proximity labeling to identify cell cycle-specific interaction partners

  • Quantitative proteomics to measure changes in the CDC37L1 interactome across cell cycle phases

• Functional genomics screens:

  • CRISPR/Cas9 screens to identify synthetic interactions between CDC37L1 and cell cycle regulators

  • Research has already established CDC37L1's impact on CDK6 expression in gastric cancer cells

• Cell cycle synchronization studies:

  • Examine CDC37L1 expression and function in synchronized cell populations

  • Flow cytometry analysis combined with CDC37L1 antibody staining to correlate with cell cycle phases

  • Flow cytometry has revealed that CDC37L1 knockdown leads to increased cells in S phase, an effect inhibited by CDK4/6 inhibitor Palbociclib

• CDK activity assays:

  • In vitro kinase assays to assess CDC37L1's impact on CDK activity

  • Phospho-specific antibodies to measure CDK substrate phosphorylation

These advanced approaches are revealing CDC37L1's mechanistic role in regulating cell proliferation through modulation of CDK6 and potentially other cell cycle proteins.