CYP40 Antibody

Structural and Functional Properties of CYP40

CyP40 belongs to the immunophilin family and contains two characteristic domains: the C-terminal tetratricopeptide repeats (TPR) and the N-terminal peptidyl prolyl cis/trans isomerase (PPIase) domain, separated by approximately 30 amino acids. The TPR repeats serve as interaction surfaces necessary for binding to Hsp90, while the PPIase domain catalyzes prolyl isomerization during protein folding processes . Human CyP40 has a protein mass of approximately 60.1 kilodaltons with two identified isoforms .

Cellular Localization and Tissue Distribution

Unlike some cyclophilins that have specific subcellular targeting, CyP40 appears to be present in both cytoplasm and nucleus . Its expression is ubiquitous across most human tissues, with notable expression in the testis, placenta, and adrenal gland, but lower expression in lung tissue . This distribution pattern has important implications for experimental design when targeting CyP40 in specific tissue contexts.

What applications are most suitable for CyP40 antibodies?

CyP40 antibodies are versatile tools applicable across multiple experimental techniques. Western blot represents the most common application, allowing researchers to detect and quantify CyP40 protein expression levels in cell and tissue lysates. Additionally, ELISA and immunohistochemistry are frequently employed techniques for CyP40 detection . When selecting an antibody, consideration should be given to the specific application requirements, with validation for each intended use being essential for reliable results.

For immunohistochemistry applications, antigen retrieval optimization is particularly important as CyP40's association with protein complexes may mask epitopes. Heat-induced epitope retrieval in citrate buffer (pH 6.0) has demonstrated effective results in multiple published protocols.

How should researchers validate CyP40 antibody specificity?

Validation of CyP40 antibodies requires multiple approaches to ensure specificity:

• Positive controls using tissues with known high CyP40 expression (adrenal, testis, placenta)

• Negative controls utilizing tissues with minimal expression or CyP40 knockdown models

• Peptide competition assays to confirm epitope specificity

• Western blot analysis to confirm detection of the correct molecular weight band (approximately 40 kDa)

• Cross-validation with multiple antibodies targeting different epitopes of CyP40

The tandem affinity purification approach has proven effective for validating protein interactions with CyP40, helping to distinguish specific from non-specific binding patterns .

What are the key considerations for sample preparation when working with CyP40 antibodies?

Effective sample preparation for CyP40 detection requires careful attention to several factors:

• Lysis buffer composition: Use buffers containing protease inhibitors (1 mM PMSF, 2 μg/ml leupeptin, and complete protease inhibitor cocktail) to prevent degradation

• Sample handling: Multiple freeze/thaw cycles (four cycles recommended) can improve extraction efficiency from cellular compartments

• Subcellular fractionation: Consider separate isolation of nuclear and cytoplasmic fractions due to CyP40's dual localization

• Preservation of protein-protein interactions: Mild detergent conditions if studying CyP40 complexes

• Denaturing conditions: More stringent lysis conditions may be needed to fully extract CyP40 from tight complexes with Hsp90

The cellular distribution of CyP40 necessitates careful sample preparation to ensure comprehensive extraction and accurate analysis of protein levels.

How can researchers effectively distinguish between CyP40 and other cyclophilin family members?

Distinguishing CyP40 from other cyclophilin family members (CyPA, CyPB, CyPC, CyPD, hCyP33) requires strategic experimental design:

• Antibody selection: Choose antibodies targeting unique regions, particularly the TPR domains that are absent in other cyclophilins

• Molecular weight discrimination: CyP40 (40 kDa) versus smaller cyclophilins (e.g., CyPA at 18 kDa)

• Subcellular localization controls: Utilize the differential localization patterns (CyPB and CyPC in endoplasmic reticulum, CyPD in mitochondria, hCyP33 in nucleus)

• Expression system validation: Express recombinant tagged versions of multiple cyclophilins to verify antibody specificity

The amino acid sequence homology between cyclophilins necessitates careful antibody selection and experimental controls to ensure specific detection of CyP40 versus related family members.

What strategies should be employed when studying CyP40 protein interactions?

Investigating CyP40 protein interactions requires sophisticated approaches:

• Tandem affinity purification: Effective for identifying novel interacting proteins as demonstrated with identified partners including RACK1, Ku70, RPS3, and NF45

• Co-immunoprecipitation validation: Essential for confirming direct protein interactions across different expression systems (bacterial, rabbit reticulocyte lysate, mammalian cells)

• Crosslinking approaches: Consider mild crosslinking to stabilize transient interactions

• Competition studies: Use cyclosporin A to investigate CyP40-dependent interactions, as it inhibits the peptidyl-prolyl isomerase activity with an IC50 of approximately 60 nM in yeast Cyp40

• Domain-specific mutants: Generate TPR domain or PPIase domain mutants to map interaction sites

The established interaction between CyP40 and Hsp90 provides an excellent positive control for interaction studies, as this association has been consistently demonstrated across species from yeast to humans .

How does CyP40 expression change under different cellular stress conditions?

CyP40 expression displays dynamic regulation under various stress conditions that should be considered in experimental design:

• Heat shock: Induces 3-4 fold increase in expression of the yeast Cyp40 homolog

• Hypoxia: CyP40 influences HIF-1α stability through interaction with RACK1, suggesting hypoxia-responsive regulation

• Calcium fluctuations: S100 proteins interact with CyP40 only in the presence of calcium, indicating calcium-dependent regulatory mechanisms

When designing experiments investigating CyP40 function under stress conditions, researchers should account for these expression changes and potential shifts in interaction partners that may occur in response to specific cellular stressors.

What are the optimal techniques for quantifying CyP40 levels in biological samples?

Quantitative assessment of CyP40 requires method-specific optimizations:

For absolute quantification, recombinant CyP40 standard curves should be generated using the same antibody and detection system as experimental samples.

How can researchers effectively design CyP40 knockdown or knockout experiments?

Strategic approaches for CyP40 functional studies include:

• shRNA knockdown: Demonstrated effective with plasmids containing shRNA specific to CyP40 (targeting NM_005038), resulting in approximately 95% reduction in cellular CyP40 content

• CRISPR-Cas9 knockout: Target conserved exons encoding the PPIase domain

• Functional rescue experiments: Re-express CyP40 in knockdown cells to verify phenotype specificity

• Domain-specific mutants: Generate PPIase-dead or TPR-deficient mutants to dissect domain-specific functions

• Cyclophilin inhibitors: Use cyclosporin A as a pharmacological approach, noting it affects multiple cyclophilins

Interestingly, complete knockout of the Cyp40 homolog in yeast resulted in viable strains with normal growth at both standard and elevated temperatures, suggesting potential functional redundancy that should be considered when interpreting knockout phenotypes in other systems .

What considerations are important when studying CyP40's role in transcriptional regulation?

CyP40's involvement in transcriptional processes requires specialized experimental approaches:

• Chromatin immunoprecipitation: Determine if CyP40 associates with specific genomic regions

• Reporter assays: Monitor effects on transcriptional activity (e.g., HRE-dependent luciferase activity as demonstrated with RACK1 and CyP40)

• Protein-protein interaction studies with transcription factors: Investigate associations with NF45, which is involved in IL-2 gene regulation

• Nuclear-cytoplasmic fractionation: Quantify nuclear translocation under different conditions

• Transcription factor complex analysis: Study CyP40's influence on complexes like those containing Ku70 and NF45

Research has demonstrated that RACK1 suppresses cobalt chloride-induced, HRE-dependent luciferase activity in MCF-7 cells but not in cells with reduced CyP40 expression, indicating CyP40's importance in certain transcriptional regulatory pathways .