Cyclophilin-E Antibody

What is Cyclophilin-E and why is it important in research?

Cyclophilin-E (also known as CyPE, Cyclophilin 33, CyP33, PPIase E, or Rotamase E) is a crucial nuclear RNA-binding protein involved in significant cellular processes. It features a unique structure with an N-terminal RNA binding domain and a C-terminal cyclophilin domain, allowing specific interactions with mRNA. This structural configuration enhances its peptidyl-prolyl isomerase activity, facilitating proper protein folding and gene expression regulation .

Cyclophilin-E is particularly important in research because it binds to the third PHD zinc finger domain of Mixed Lineage Leukemia (MLL) protein, influencing transcriptional regulation of target genes including HoxC8 and HoxC9. Its overexpression can inhibit transcription of these genes by promoting histone deacetylase 1 binding to MLL repression domain . Additionally, it functions in pre-mRNA splicing as a component of the spliceosome and demonstrates preference for single-stranded RNA molecules with poly-A and poly-U stretches .

What types of Cyclophilin-E antibodies are available for research applications?

Several types of Cyclophilin-E antibodies are available, each with specific characteristics suitable for different research applications:

The choice of antibody depends on the specific research application, species of interest, and detection method required.

How does Cyclophilin-E differ from other cyclophilins like Cyclophilin A?

Cyclophilin-E differs significantly from other cyclophilins such as Cyclophilin A in structure, cellular localization, and function:

• Structure and Domains: Cyclophilin-E uniquely contains both an RNA-binding domain (RRM) at its N-terminus and a cyclophilin domain at its C-terminus . In contrast, Cyclophilin A is smaller (~18 kDa) and lacks the RNA-binding domain .

• Cellular Localization: Cyclophilin-E is predominantly nuclear and participates in nuclear processes like pre-mRNA splicing . Cyclophilin A is ubiquitously distributed intracellularly and can be secreted by cells in response to inflammatory stimuli .

• Functional Roles: While both possess peptidyl-prolyl isomerase activity, Cyclophilin-E specifically regulates transcription through interactions with MLL protein and demonstrates antiviral properties against influenza virus by interacting with viral nucleoprotein . Cyclophilin A interacts with HIV proteins and is necessary for infectious HIV virion formation .

• Research Applications: Antibodies against these different cyclophilins target distinct biological processes and pathways, making them valuable for specific research questions related to either nuclear gene regulation (Cyclophilin-E) or cytoplasmic and secretory pathways (Cyclophilin A).

What are the optimal conditions for using Cyclophilin-E antibodies in Western blotting experiments?

Optimizing Western blotting with Cyclophilin-E antibodies requires careful attention to several parameters:

Sample Preparation and Loading:

• Use fresh lysates when possible, with complete protease inhibitor cocktails

• For Cyclophilin-E detection, a concentration range of 0.04-0.4 μg/ml is recommended for polyclonal antibodies

• Include positive controls such as lysates from cells known to express Cyclophilin-E (HEK293T cells with PPIE overexpression work well)

Blocking and Antibody Incubation:

• For monoclonal antibodies like clone 9E18, use standard blocking solutions (5% non-fat milk or BSA)

• Primary antibody incubation is typically performed overnight at 4°C

• For the rabbit polyclonal antibody (NBP1-85365), optimal dilution ranges from 1:50 to 1:200 for immunohistochemistry and 0.04-0.4 μg/ml for Western blotting

Validation Controls:

• Include a vector-only transfected control alongside PPIE overexpression lysate to confirm specificity

• The Cyclophilin-E protein should be detected at approximately 33 kDa

• For tagged proteins, adjust the expected molecular weight accordingly (e.g., C-terminal myc-DDK tag adds 3.1 kDa)

Western blot analysis has successfully demonstrated specificity of anti-Cyclophilin-E antibodies by comparing control (vector-only transfected HEK293T lysate) with PPIE overexpression lysate , confirming the antibody's ability to specifically detect the target protein.

How can I optimize immunohistochemistry protocols for Cyclophilin-E detection in tissue samples?

Successful immunohistochemical detection of Cyclophilin-E requires careful optimization:

Tissue Preparation and Antigen Retrieval:

• For paraffin-embedded sections, heat-induced epitope retrieval (HIER) at pH 6 is specifically recommended

• Complete deparaffinization and rehydration are essential for consistent results

Antibody Selection and Dilution:

• For polyclonal antibodies like NBP1-85365, optimal dilution ranges from 1:50 to 1:200

• Incubation times may require optimization (typically overnight at 4°C or 1-2 hours at room temperature)

Signal Detection and Interpretation:

• Cyclophilin-E typically shows strong nuclear positivity, as demonstrated in human cerebellum samples

• Include positive tissue controls with known Cyclophilin-E expression

• Consider dual staining with markers of cellular compartments to confirm localization

When properly optimized, immunohistochemistry with anti-Cyclophilin-E antibody reveals strong nuclear localization pattern, consistent with its known role in nuclear processes such as pre-mRNA splicing and transcriptional regulation .

What controls should be included when using Cyclophilin-E antibodies to ensure experimental validity?

Rigorous controls are essential for reliable interpretation of experiments using Cyclophilin-E antibodies:

Positive Controls:

• Lysates or tissues with confirmed Cyclophilin-E expression

• Recombinant Cyclophilin-E protein can serve as a positive control in Western blots

• HEK293T cells with PPIE overexpression provide an excellent positive control

Negative Controls:

• Primary antibody omission to assess non-specific binding of secondary antibodies

• Isotype-matched control antibodies (especially important for monoclonal antibodies like 9E18)

• For siRNA experiments, non-targeting siRNA controls should be used as demonstrated in influenza virus studies

Specificity Controls:

• Comparison between wild-type and Cyclophilin-E knockout/knockdown samples

• Pre-adsorption with recombinant Cyclophilin-E protein

• Testing on multiple expression systems and cell lines to ensure consistent results

Functional Validation:

• For studies investigating Cyclophilin-E's PPIase activity, include wild-type protein and catalytically inactive mutants

• When studying protein-protein interactions (like with MLL), include domain deletion mutants to confirm specificity of binding regions

In published research, specificity of anti-Cyclophilin-E antibodies has been validated using overexpression systems, comparing vector-only controls with Cyclophilin-E-expressing cells, and through domain deletion studies that confirm functional specificity .

How can Cyclophilin-E antibodies be used to investigate its role in viral infections?

Cyclophilin-E has emerged as an important host factor in viral infections, particularly influenza virus. Antibodies against Cyclophilin-E can be strategically employed to investigate these interactions:

Co-Immunoprecipitation Studies:

• Anti-Cyclophilin-E antibodies have been used to demonstrate direct interaction between Cyclophilin-E and viral proteins

• In influenza research, cell lysates immunoblotted with anti-Myc or anti-FLAG antibodies confirmed expression of proteins of interest in 293T cells

• These studies revealed that Cyclophilin-E functions as a negative regulator to influenza virus by impairing the formation of viral ribonucleoprotein (vRNP)

Functional Validation Through Knockdown/Overexpression:

• Knockdown of endogenous Cyclophilin-E has been shown to favor influenza virus replication

• Conversely, overexpression of Cyclophilin-E decreased viral nucleoprotein levels and reduced virus titers approximately twofold compared to control cells

• Importantly, this antiviral effect was dependent on Cyclophilin-E binding to viral nucleoprotein, as demonstrated using the Cyclophilin-E Δ137–186 deletion mutant

Methodological Approach:

• Perform co-immunoprecipitation using anti-Cyclophilin-E antibodies to pull down viral protein complexes

• Use Western blotting with anti-Cyclophilin-E antibodies to monitor expression levels in knockdown or overexpression experiments

• Complement with immunofluorescence studies to visualize subcellular localization during infection

• Utilize domain mutants to map specific interaction regions between Cyclophilin-E and viral proteins

These approaches have established Cyclophilin-E as a host restriction factor that targets viral nucleoprotein functions, providing a potential target for antiviral strategies .

What are the best approaches for studying Cyclophilin-E's interaction with the MLL protein complex?

Studying Cyclophilin-E's interaction with the MLL (Mixed Lineage Leukemia) protein complex requires sophisticated methodological approaches:

Co-Immunoprecipitation and Pulldown Assays:

• Anti-Cyclophilin-E antibodies can be used to precipitate native protein complexes containing MLL

• These experiments have revealed that Cyclophilin-E binds to the third PHD zinc finger domain of MLL protein

• This interaction influences transcriptional regulation of target genes including HoxC8 and HoxC9

Functional Analysis Through Gene Expression Studies:

• Cyclophilin-E overexpression inhibits transcription of MLL target genes by promoting histone deacetylase 1 binding to MLL repression domain

• Researchers can use anti-Cyclophilin-E antibodies in chromatin immunoprecipitation (ChIP) assays to detect recruitment to specific genomic loci

Domain Mapping and Mutational Analysis:

• The peptidyl-prolyl isomerase activity of Cyclophilin-E is required for inhibition of KMT2A (MLL) activity

• Strategic use of domain-specific antibodies or epitope-tagged constructs can help map the specific regions involved in these interactions

Methodological Workflow:

• Perform co-IP experiments using anti-Cyclophilin-E antibodies to confirm interaction with MLL complex components

• Use ChIP with anti-Cyclophilin-E to identify genomic binding sites

• Combine with gene expression analysis after Cyclophilin-E knockdown or overexpression

• Validate with domain mutants and functional readouts of MLL activity

These approaches have established Cyclophilin-E as an important regulator of MLL function, with significant implications for understanding gene regulation in developmental processes and diseases .

How can Cyclophilin-E antibodies be utilized in studies of pre-mRNA splicing mechanisms?

Cyclophilin-E's involvement in pre-mRNA splicing as a spliceosome component makes its antibodies valuable tools for investigating this fundamental cellular process:

Spliceosome Complex Analysis:

• Anti-Cyclophilin-E antibodies can immunoprecipitate spliceosome complexes for proteomic analysis

• This approach has confirmed Cyclophilin-E as a component of the spliceosome involved in pre-mRNA splicing

RNA-Binding Studies:

• Cyclophilin-E combines RNA-binding and PPIase activities, with preference for single-stranded RNA molecules containing poly-A and poly-U stretches

• RNA immunoprecipitation (RIP) with anti-Cyclophilin-E antibodies can identify bound RNA species

• These experiments suggest Cyclophilin-E binds to the poly(A)-region in the 3'-UTR of mRNA molecules

Functional Splicing Assays:

• Depletion or inhibition of Cyclophilin-E can be monitored using Western blotting with anti-Cyclophilin-E antibodies

• Effects on splicing can be assessed using minigene reporters or RNA-seq to detect alterations in splicing patterns

Methodological Strategy:

• Utilize anti-Cyclophilin-E antibodies for immunofluorescence to co-localize with other splicing factors

• Perform RIP-seq to identify RNA targets bound by Cyclophilin-E

• Use knockdown/overexpression approaches with splicing-sensitive reporters

• Complement with in vitro splicing assays using immunodepleted nuclear extracts

These approaches provide insights into how Cyclophilin-E's dual RNA-binding and PPIase activities contribute to proper pre-mRNA processing, with implications for understanding splicing regulation in normal and disease states .

What are common issues encountered when using Cyclophilin-E antibodies in Western blotting, and how can they be resolved?

Researchers using Cyclophilin-E antibodies in Western blotting may encounter several challenges:

High Background or Non-specific Bands:

• Problem: Multiple bands or high background obscuring the specific Cyclophilin-E signal

• Solution: Increase blocking time/concentration, optimize antibody dilution (0.04-0.4 μg/ml recommended for polyclonal antibodies) , use more stringent washing conditions, and consider alternative blocking reagents

Weak or No Signal:

• Problem: Failure to detect Cyclophilin-E despite predicted expression

• Solution: Check sample preparation (fresh lysates with protease inhibitors), increase protein loading, optimize transfer conditions for proteins in the 33 kDa range, and consider enhanced chemiluminescence detection systems

Inconsistent Results Between Samples:

• Problem: Variable detection across similar samples

• Solution: Standardize protein quantification methods, ensure equal loading using housekeeping controls, and maintain consistent sample preparation protocols

Band Size Discrepancies:

• Problem: Detected band at unexpected molecular weight

• Solution: Cyclophilin-E should appear at approximately 33 kDa; post-translational modifications or splice variants may alter migration. Include positive controls like PPIE-overexpressing HEK293T cells , and consider the addition of tags when using recombinant systems (e.g., myc-DDK tag adds 3.1 kDa)

Low Reproducibility:

• Problem: Results vary between experiments

• Solution: Standardize protocols, use the same antibody lot when possible, and include consistent positive and negative controls in each experiment

Successfully addressing these issues enables reliable detection of Cyclophilin-E, as demonstrated in published research comparing control and PPIE-overexpressing cell lysates .

How should researchers interpret contradictory results when comparing different anti-Cyclophilin-E antibodies?

Contradictory results between different anti-Cyclophilin-E antibodies require systematic analysis:

Epitope Differences:

• Different antibodies target distinct epitopes on Cyclophilin-E

• The monoclonal antibody 9E18 recognizes specific epitopes that may be masked in certain experimental conditions

• Polyclonal antibodies like NBP1-85365 were developed against a specific recombinant protein fragment (amino acids: EEVDDKVLHAAFIPFGDITDIQIPLDYETEKHRGFAFVEFELAEDAAAAIDNMNESELFGRTIRVNLAKPMRIKEGSSRPVWSDDDWLKKF)

Resolution Strategy:

• Comparative Analysis: Test multiple antibodies side-by-side on identical samples

• Validation Controls: Include Cyclophilin-E overexpression, knockdown, and knockout samples

• Alternative Detection Methods: Complement antibody-based detection with mass spectrometry or RNA expression data

• Domain-Specific Analysis: Consider whether results differ due to detection of specific protein domains, isoforms, or post-translational modifications

Interpretation Framework:

• Consistent results across multiple antibodies provide stronger evidence

• Discrepancies may reveal biologically significant information about protein conformation, interactions, or modifications

• Results should be interpreted in the context of the specific experimental system and conditions

When analyzing contradictory results, researchers should consider the specific properties of each antibody, including clonality, host species, and the immunogen used for development , as these factors significantly impact detection patterns and specificity.

What approaches can help troubleshoot non-specific staining in immunohistochemistry with Cyclophilin-E antibodies?

Non-specific staining is a common challenge in immunohistochemistry that requires systematic troubleshooting:

Optimizing Blocking Conditions:

• Increase blocking time or concentration

• Test alternative blocking agents (BSA, normal serum, commercial blocking reagents)

• For polyclonal antibodies like NBP1-85365, thorough blocking is particularly important

Antigen Retrieval Optimization:

• For paraffin sections, HIER pH 6 retrieval is specifically recommended for Cyclophilin-E detection

• Adjust retrieval time and temperature based on tissue type and fixation conditions

Antibody Dilution Series:

• Test a range of antibody dilutions (1:50 - 1:200 recommended for NBP1-85365)

• Perform parallel staining with serial dilutions to identify optimal signal-to-noise ratio

Controls to Identify Sources of Non-specificity:

• Primary antibody omission

• Isotype controls

• Pre-absorption with recombinant Cyclophilin-E protein

• Comparison with known positive samples (human cerebellum shows strong nuclear positivity)

Signal Development Optimization:

• Reduce substrate development time

• Use alternative detection systems

• Consider fluorescent detection for better signal discrimination

Interpretation Guidelines:

• Cyclophilin-E shows predominantly nuclear localization

• Cytoplasmic staining may represent non-specific binding or cross-reactivity

• Pattern should be consistent with known biology (nuclear localization for transcription/splicing functions)

Proper optimization typically yields clear nuclear staining in appropriate tissues, as demonstrated with anti-Cyclophilin-E antibody in human cerebellum samples .

How can Cyclophilin-E antibodies be integrated with new technologies for studying protein-RNA interactions?

Cutting-edge research on Cyclophilin-E's RNA-binding functions benefits from integrating antibodies with emerging technologies:

CLIP-Seq Applications:

• Cross-linking immunoprecipitation sequencing (CLIP-seq) using anti-Cyclophilin-E antibodies can map RNA binding sites with nucleotide resolution

• This approach extends previous findings that Cyclophilin-E binds single-stranded RNA with preference for poly-A and poly-U stretches

• CLIP-seq can identify the precise RNA motifs and structures recognized by Cyclophilin-E's RNA-binding domain

Proximity Labeling Approaches:

• BioID or APEX2 fusions with Cyclophilin-E can identify proteins in proximity during RNA processing

• Anti-Cyclophilin-E antibodies verify expression and localization of fusion proteins

• These methods provide spatial context for Cyclophilin-E function in spliceosomes and transcriptional complexes

Single-Molecule Imaging:

• Anti-Cyclophilin-E antibodies conjugated to fluorophores enable tracking of individual molecules

• Combined with labeled RNA, this approach can visualize RNA-protein interactions in real-time

• These techniques help understand the dynamics of Cyclophilin-E's binding to target RNAs

Cryo-EM Structural Analysis:

• Antibody fragments can facilitate structure determination of Cyclophilin-E complexes

• This approach may reveal conformational changes associated with RNA binding and isomerization

• Structural insights complement functional data on Cyclophilin-E's dual domains

These integrated approaches extend beyond traditional applications, providing deeper insights into how Cyclophilin-E's RNA-binding and PPIase activities coordinate to regulate splicing and gene expression .

What is the current understanding of Cyclophilin-E's role in disease pathogenesis, and how can antibodies advance this research?

Emerging research suggests Cyclophilin-E's involvement in several disease processes, with antibodies serving as critical tools for investigation:

Viral Infections:

• Cyclophilin-E functions as a negative regulator of influenza virus replication

• Anti-Cyclophilin-E antibodies have demonstrated that CypE interacts with viral nucleoprotein

• Overexpression studies using tagged Cyclophilin-E (detected with anti-Myc) showed decreased viral replication and approximately twofold reduction in virus titer

• Importantly, this antiviral effect requires binding to nucleoprotein, as the CypE Δ137–186 deletion mutant loses this activity

Transcriptional Dysregulation:

• Cyclophilin-E regulates gene expression through interaction with the MLL protein, influencing HoxC8 and HoxC9 genes

• Antibodies enable chromatin immunoprecipitation to map genomic binding sites

• These approaches may reveal Cyclophilin-E's involvement in developmental disorders or cancers associated with MLL dysregulation

Splicing-Related Disorders:

• As a spliceosome component , Cyclophilin-E could contribute to splicing-related diseases

• Anti-Cyclophilin-E antibodies can help identify aberrant spliceosome composition or localization

• Immunoprecipitation followed by RNA analysis may reveal altered RNA binding in disease states

Research Applications:

• Use antibodies to assess Cyclophilin-E expression levels in disease tissues

• Perform co-immunoprecipitation to identify altered protein interactions in pathological states

• Develop therapeutic strategies targeting Cyclophilin-E's PPIase activity or protein interactions

• Employ domain-specific antibodies to distinguish functional activities in different contexts

This research direction may identify Cyclophilin-E as a therapeutic target or biomarker for viral infections and potentially other diseases involving RNA processing dysregulation .

What methodological considerations are important when using Cyclophilin-E antibodies in multi-omics research approaches?

Integrating Cyclophilin-E antibodies into multi-omics workflows requires careful methodological considerations:

Immunoprecipitation for Proteomics:

• Anti-Cyclophilin-E antibodies can enrich protein complexes for mass spectrometry analysis

• Consider cross-linking approaches to capture transient interactions

• Compare results from multiple antibodies targeting different epitopes

• Include appropriate controls (IgG, isotype controls) for accurate interaction identification

ChIP-Seq and RNA-Binding Analysis:

• Optimize chromatin immunoprecipitation conditions for Cyclophilin-E's nuclear localization

• Validate antibody specificity in both IP and Western blot before proceeding to sequencing

• Consider sequential ChIP to identify co-occupancy with other factors

• For RNA studies, RIP-seq or CLIP-seq require highly specific antibodies with minimal background

Integration with Transcriptomics:

• Correlate Cyclophilin-E binding sites with transcriptome changes after manipulation

• Compare RNA-seq data from wild-type and Cyclophilin-E knockdown/knockout systems

• Analyze alternative splicing events that may depend on Cyclophilin-E's spliceosome function

Data Analysis Considerations:

• Account for antibody efficiency/bias in computational analysis pipelines

• Use appropriate normalization strategies when comparing datasets generated with different antibodies

• Integrate results from complementary approaches (e.g., proteomics, genomics, transcriptomics)

• Consider biological replicates to ensure reproducibility

Quality Control Metrics:

• Verify antibody specificity before large-scale experiments

• Include spike-in controls for quantitative analyses

• Document lot-to-lot variation that may affect multi-omics data comparability

• Validate key findings with orthogonal methods

These considerations enable robust multi-omics research that can comprehensively characterize Cyclophilin-E's diverse functions in pre-mRNA splicing , transcriptional regulation , and antiviral response .