CPSF160 Antibody

What is CPSF160 and why is it important in RNA processing research?

CPSF160 (also known as CPSF1) is the largest subunit of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex that plays a crucial role in pre-mRNA 3'-end formation. It recognizes the canonical AAUAAA hexamer signal sequence and interacts with poly(A) polymerase and other factors to facilitate cleavage and poly(A) addition . CPSF160 is particularly important because it functions in the RNA recognition step of the polyadenylation reaction . Beyond its canonical role in mRNA processing, CPSF160 may also play roles in developmental processes, as studies suggest it contributes to eye morphogenesis and the development of retinal ganglion cell projections to the midbrain . As a key component of the mRNA processing machinery, studying CPSF160 provides insights into fundamental cellular mechanisms that regulate gene expression.

Which experimental techniques are compatible with CPSF160 antibodies?

CPSF160 antibodies have been validated for multiple experimental applications in molecular biology research:

• Immunoprecipitation (IP): CPSF160 antibodies can effectively pull down both the protein itself and its associated complex members .

• Western Blotting (WB): Commercially available antibodies have been validated for detecting CPSF160 in protein extracts .

• Immunohistochemistry on Paraffin-embedded sections (IHC-P): CPSF160 can be detected in fixed tissue samples, making it possible to study its expression patterns in different tissues and pathological conditions .

• RNA Immunoprecipitation: CPSF160 antibodies can be used to study protein-RNA interactions, as demonstrated by their ability to co-immunoprecipitate RNA containing polyadenylation signals .

• Co-immunoprecipitation: These antibodies can pull down other CPSF complex components, enabling studies of protein-protein interactions within the 3' end processing machinery .

How should researchers validate CPSF160 antibody specificity for their experiments?

Validating antibody specificity is crucial for reliable experimental outcomes. For CPSF160 antibodies, consider the following validation approaches:

• Western Blot Analysis: Confirm the antibody detects a single band of approximately 160 kDa in your experimental system. Compare results across different cell lines or tissues to verify consistent detection patterns.

• Knockdown/Knockout Controls: Perform siRNA knockdown or CRISPR-Cas9 knockout of CPSF160 and confirm reduced or absent signal by Western blot or immunostaining.

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide (if available) before application to your sample. The specific signal should be blocked or significantly reduced.

• Cross-validation with Multiple Antibodies: Use antibodies raised against different epitopes of CPSF160 to confirm your findings.

• Immunoprecipitation-Mass Spectrometry: After IP with anti-CPSF160 antibody, perform mass spectrometry to confirm the presence of CPSF160 and expected interacting partners like CPSF100, CPSF73, and Fip1 .

What are the optimal conditions for immunoprecipitation with CPSF160 antibodies?

Based on published protocols, the following considerations can optimize CPSF160 immunoprecipitation:

• Lysis Buffer Composition: Use a buffer containing 20 mM HEPES, pH 7.4, 130 mM NaCl, 2 mM EDTA, and 0.25% Triton X-100 with protease inhibitors . This composition preserves protein-protein interactions while effectively solubilizing nuclear proteins.

• Incubation Time and Temperature: Lyse cells for 30 minutes at 4°C and clarify by centrifugation at 13,000 × g .

• Antibody-to-Protein Ratio: Typically, 2-5 μg of antibody per 500 μg of total protein provides good results for CPSF160 IP.

• Controls: Include isotype-matched IgG controls to identify non-specific binding .

• RNase Treatment: To determine if interactions are RNA-dependent, treat a portion of your samples with RNase A. CPSF160's interaction with Fip1 has been shown to be RNA-independent, persisting after RNase treatment .

• Detection of Interacting Partners: When probing for CPSF complex members, be aware that some proteins like Fip1 may appear as multiple bands (around 70 kDa) in Western blots, which is the typical pattern for this protein .

How can CPSF160 antibodies be used to investigate protein complexes in polyadenylation?

CPSF160 antibodies are valuable tools for investigating the composition and dynamics of polyadenylation complexes:

• Sequential Immunoprecipitation: To isolate specific subcomplexes, perform sequential IP first with anti-CPSF160 antibody followed by another component. This approach has revealed that CPSF160, CPSF30, hFip1, and WDR33 form a "mammalian polyadenylation specificity factor" (mPSF) subcomplex that is active in AAUAAA-dependent polyadenylation .

• Analysis of Novel Interactions: Co-immunoprecipitation experiments with CPSF160 antibodies have revealed interactions not only within the CPSF complex but also with other 3' processing factors like CstF64 .

• Stoichiometry Analysis: Quantitative analysis of immunoprecipitated complexes can provide insights into the relative abundance of different subunits. Research has shown that when CPSF160 and WDR33 are present in equal amounts, hFip1 can be present at a 1.8-fold excess, and CPSF30 at a 2.5-3.1-fold excess .

• Functional Complex Reconstitution: By immunodepleting CPSF160 and its associated proteins from nuclear extracts, researchers can assess the functional impact on in vitro cleavage and polyadenylation assays. Adding back purified components allows for determination of which factors are necessary and sufficient for activity .

What methods can be used to study CPSF160-RNA interactions using specific antibodies?

Several techniques can be employed to investigate CPSF160-RNA interactions:

• RNA Immunoprecipitation (RIP): This technique allows identification of RNAs bound to CPSF160 in vivo. Using anti-CPSF160 antibodies, researchers have demonstrated that CPSF160 associates with RNAs containing polyadenylation signals, such as the SV40 late polyadenylation signal .

• UV Cross-linking Followed by Immunoprecipitation: By UV-irradiating cells prior to lysis and immunoprecipitation with CPSF160 antibodies, researchers can identify direct RNA-protein contacts. This approach has shown that while CPSF160 is essential for RNA binding, WDR33 appears to make direct contact with the AAUAAA motif .

• In Vitro Binding Assays: Purified CPSF160 (often as part of a reconstituted complex) can be tested for binding to labeled RNA substrates containing various polyadenylation signals. Antibodies can then be used to detect or immunoprecipitate the complexes formed.

• CLIP-seq (Cross-linking Immunoprecipitation followed by Sequencing): This technique combines UV cross-linking, immunoprecipitation with CPSF160 antibodies, and high-throughput sequencing to map RNA binding sites genome-wide.

• Structure-Function Analysis: By combining RNA binding assays with immunoprecipitation of CPSF160 mutants, researchers can map the RNA-binding domains and understand the structural basis of substrate recognition.

What are common issues when working with CPSF160 antibodies and how can they be resolved?

Several challenges may arise when working with CPSF160 antibodies, with corresponding solutions:

• High Background in Immunoprecipitation:

  • Increase washing stringency with higher salt concentrations (up to 300 mM NaCl)

  • Use a preclearing step with protein A/G beads before adding the antibody

  • Include competitors for non-specific interactions (e.g., 0.1-0.5% BSA)

• Poor Recovery in Co-immunoprecipitation:

  • Optimize crosslinking conditions if using formaldehyde crosslinking

  • Use gentler lysis buffers (reduce detergent concentration)

  • Ensure antibodies recognize native protein conformations, not just denatured epitopes

• Inconsistent Results in RNA-IP:

  • Include RNase inhibitors in all buffers

  • Optimize UV cross-linking times for RNA-protein interactions

  • Consider the impact of different salt concentrations on RNA-protein complex stability

• Multiple Bands in Western Blot:

  • Verify if bands represent isoforms, post-translational modifications, or degradation products

  • Use freshly prepared samples with protease inhibitors

  • For closely related family members, validate with knockout controls

• Poor Signal in IHC-P:

  • Optimize antigen retrieval methods (citrate buffer vs. EDTA buffer at different pH values)

  • Test different antibody concentrations and incubation times

  • Consider signal amplification systems for low-abundance targets

How can researchers quantitatively assess CPSF160 antibody performance across different applications?

Quantitative assessment of antibody performance is crucial for reproducible research:

• Titration Curves: Perform serial dilutions of antibody to determine the optimal concentration for each application that maximizes specific signal while minimizing background.

• Signal-to-Noise Ratio Calculation: For imaging applications, calculate the ratio of specific signal to background using image analysis software to objectively compare antibody performance.

• Recovery Rate in IP: Quantify the percentage of target protein recovered through immunoprecipitation compared to the input sample using densitometry of Western blots.

• Reproducibility Analysis: Perform technical and biological replicates to calculate coefficient of variation (%CV) for key measurements.

• Cross-Platform Validation: Compare results obtained with the same antibody across different detection methods (e.g., Western blot vs. immunofluorescence vs. ELISA).

• Positive and Negative Controls: Include samples with known high and low/no expression of CPSF160 to establish the dynamic range of detection.

How can CPSF160 antibodies be used to investigate alternative polyadenylation in disease models?

Alternative polyadenylation (APA) is increasingly recognized as a mechanism dysregulated in various diseases. CPSF160 antibodies can be valuable tools in this research:

• ChIP-seq for CPSF160 Occupancy: Map genome-wide binding sites of CPSF160 in normal versus disease samples to identify differential occupancy at polyadenylation sites.

• IP-RT-PCR for Isoform Analysis: Use CPSF160 immunoprecipitation followed by RT-PCR with isoform-specific primers to detect shifts in poly(A) site usage in specific transcripts.

• Proximity Ligation Assays: Combine CPSF160 antibodies with antibodies against disease-relevant factors to detect altered protein-protein interactions within the polyadenylation machinery.

• Functional Recovery Experiments: In cells depleted of endogenous CPSF160, express wild-type or mutant versions (associated with disease) and use CPSF160 antibodies to immunoprecipitate reconstituted complexes for functional testing.

• Tissue Microarray Analysis: Apply CPSF160 antibodies in IHC-P to analyze expression patterns across multiple patient samples, correlating with clinical outcomes and molecular subtypes.

Recent research suggests that CPSF1 positively regulates NSDHL by alternative polyadenylation and promotes gastric cancer progression , highlighting the relevance of these approaches for cancer research.

What are the technical considerations for analyzing CPSF160 interactions in chromatin-associated complexes?

Studying CPSF160 in the context of chromatin requires specialized approaches:

• Chromatin Fractionation: Prior to immunoprecipitation with CPSF160 antibodies, separate chromatin-bound proteins from soluble nuclear proteins to enrich for functional complexes.

• Sequential ChIP (Re-ChIP): Perform ChIP with CPSF160 antibodies followed by a second round with antibodies against chromatin modifiers or transcription factors to identify co-occupied genomic regions.

• Salt Extraction Series: Use increasing salt concentrations to extract proteins from chromatin, then perform Western blotting with CPSF160 antibodies to determine the strength of chromatin association.

• Nuclease Sensitivity: Treat chromatin with different nucleases (DNase I, micrococcal nuclease) before CPSF160 immunoprecipitation to determine if interactions are DNA-dependent or structure-dependent.

• Nascent RNA Analysis: Combine CPSF160 immunoprecipitation with nascent RNA isolation techniques to capture actively processing transcripts associated with the protein.

• Proximity-Dependent Labeling: Use techniques like BioID or APEX2 fused to CPSF160 to identify proteins in close proximity within the chromatin environment, followed by validation with co-immunoprecipitation using CPSF160 antibodies.

How can CPSF160 antibodies be integrated with other techniques to study 3' end processing dynamics?

Combining CPSF160 antibody-based methods with other techniques provides comprehensive insights:

• Live-Cell Imaging + Fixed-Cell Analysis: Use fluorescently tagged CPSF160 for live imaging of dynamics, followed by immunostaining with antibodies against endogenous CPSF160 and other factors to validate observations in fixed cells.

• Mass Spectrometry + Immunoprecipitation: Perform IP-MS with CPSF160 antibodies under different cellular conditions to identify condition-specific interaction partners, followed by targeted validation.

• CRISPR Screening + Antibody-Based Phenotyping: After CRISPR screens for genes affecting polyadenylation, use CPSF160 antibodies to assess changes in complex formation and localization.

• 3D Genomics + ChIP-seq: Combine Hi-C or related techniques with CPSF160 ChIP-seq to understand how 3D genome organization influences polyadenylation site choice.

• Computational Prediction + Experimental Validation: Use bioinformatic approaches to predict novel CPSF160 binding sites, followed by validation with CPSF160 antibody-based techniques like ChIP-qPCR or RIP-qPCR.

• Single-Cell Analysis: Adapt CPSF160 immunostaining protocols for single-cell analysis to investigate cell-to-cell variability in 3' end processing.

What are the emerging applications of CPSF160 antibodies in studying RNA processing regulation?

Several cutting-edge applications are expanding the utility of CPSF160 antibodies:

• Spatial Transcriptomics: Combine CPSF160 immunostaining with spatial transcriptomics to correlate protein localization with region-specific APA patterns in tissues.

• Liquid-Liquid Phase Separation (LLPS) Studies: Investigate if CPSF160 participates in phase-separated condensates using immunofluorescence under different cellular conditions that promote or disrupt LLPS.

• RNA Editing Connection: Explore potential crosstalk between RNA editing and polyadenylation by performing sequential IP with CPSF160 antibodies and antibodies against RNA editing factors.

• Non-canonical Functions: Investigate potential roles of CPSF160 beyond mRNA processing using CPSF160 antibodies to detect unexpected cellular localizations or interaction partners.

• Single-Molecule Imaging: Adapt CPSF160 antibodies for single-molecule techniques to observe the dynamics of individual processing events.

• Extracellular RNA Processing: Explore whether CPSF160 has roles in processing RNAs destined for extracellular vesicles using fractionation followed by immunoblotting with CPSF160 antibodies.