CPSF73-I Antibody

What is CPSF73 and why is it important to study?

CPSF73 is a component of the cleavage and polyadenylation specificity factor (CPSF) complex that plays a key role in pre-mRNA 3'-end formation. It recognizes the AAUAAA signal sequence and interacts with poly(A) polymerase to facilitate mRNA cleavage and polyadenylation. Without CPSF73's cleavage activity, mRNAs cannot be polyadenylated and released from transcription sites for cytoplasmic export, making it crucial for gene expression regulation . CPSF73 is also required for transcription termination defining gene boundaries, preventing transcriptional interference at downstream genes . Recent studies have associated CPSF73 activity with cancer phenotypes, positioning it as a potential biomarker and therapeutic target .

What applications are CPSF73 antibodies suitable for?

CPSF73 antibodies have been validated for multiple experimental applications including:

For optimal results, antibody concentration should be empirically determined for each application, typically starting with manufacturer recommendations (e.g., 0.04-1 μg/mL for Western blots) .

How should I validate a CPSF73 antibody for my research?

Proper validation requires multiple approaches:

• Positive control testing: Use cell lines known to express CPSF73 (e.g., HeLa, 293T, NIH3T3)

• Molecular weight verification: Confirm band appears at predicted 77 kDa

• Specificity testing: Include negative controls (e.g., isotype control antibodies)

• Cross-reactivity assessment: If working with non-human samples, verify species reactivity (human, mouse, and rat reactivity are common for commercial antibodies)

• Knockdown/knockout validation: Use siRNA or CRISPR-edited cells with reduced CPSF73 expression to confirm antibody specificity

How can I evaluate the role of CPSF73 in complex formation with other mRNA processing factors?

To investigate CPSF73's interactions within the mRNA processing complex:

• Co-immunoprecipitation approach:

  • Immunoprecipitate CPSF73 using a specific antibody (0.1-3 μg antibody per mg of lysate)

  • Perform Western blot analysis to detect co-purifying factors like symplekin, CPSF100, and CstF64

  • Include RNase A treatment controls (typically 100 μg/mL) to determine if interactions are RNA-dependent

• Triple fusion protein system:

  • Generate fusion constructs containing symplekin-CPSF73-CPSF100 to ensure stoichiometric expression

  • Compare wild-type and mutant variants to assess functional significance of specific domains

  • This approach eliminates variables related to different expression levels of individual components

• Proximity ligation assays:

  • Use pairs of antibodies against CPSF73 and potential interacting partners

  • This technique visualizes protein-protein interactions in situ with high sensitivity

How can I investigate the contribution of CPSF73's MBL motifs to its endonuclease activity?

The metallo-beta-lactamase (MBL) motifs in CPSF73 are critical for its endonuclease function. To study their importance:

• Site-directed mutagenesis approach:

  • Generate point mutations in conserved MBL motifs: H73A, D75A, H76A in motif 2 and H396A in motif B

  • Express mutant proteins in an appropriate system (e.g., HEK293 cells)

  • Compare with control mutations outside conserved motifs (e.g., S334A)

• Complementation assay methodology:

  • Use heat-inactivated nuclear extracts (typically 15 min at 50°C) that retain most processing factors but lack functional symplekin

  • Add purified wild-type or mutant CPSF73 proteins

  • Assess restoration of pre-mRNA processing activity to evaluate functional significance of specific residues

• Structural analysis:

  • Combine experimental data with structural predictions to map critical residues

  • This integrated approach provides mechanistic insights into CPSF73 function

What factors might affect CPSF73 antibody specificity in Western blot applications?

Several factors can influence specificity and should be systematically addressed:

• Sample preparation considerations:

  • CPSF73 is primarily nuclear; ensure efficient nuclear protein extraction

  • Use protease inhibitors to prevent degradation (complete cocktail recommended)

  • Include phosphatase inhibitors if investigating post-translational modifications

• Blocking and washing optimization:

  • Test different blocking agents (5% BSA vs. 5% non-fat milk)

  • Optimize primary antibody concentration (typically 0.04-1 μg/mL)

  • Include 0.05-0.1% Tween-20 in wash buffers to reduce background

• Detection system selection:

  • ECL detection systems work well with exposure times around 30 seconds

  • For weaker signals, consider enhanced sensitivity substrates or longer exposure times

• Common issues and solutions:

  • Multiple bands: May indicate degradation products or post-translational modifications

  • No signal: Check protein transfer efficiency and primary antibody concentration

  • High background: Increase washing steps or reduce antibody concentration

How can I distinguish between CPSF73 isoforms or modified forms?

To differentiate between CPSF73 variants:

• Gel system optimization:

  • Use gradient gels (4-15%) for better resolution of closely migrating forms

  • Consider Phos-tag™ gels to separate phosphorylated forms

• Immunoprecipitation followed by mass spectrometry:

  • Immunoprecipitate CPSF73 using a validated antibody

  • Analyze precipitated protein by mass spectrometry to identify modifications

  • Compare results under different cellular conditions

• Antibody selection considerations:

  • Verify the epitope recognized by the antibody (e.g., N-terminal vs. C-terminal)

  • Some antibodies may recognize specific post-translationally modified forms

How can CPSF73 antibodies be used to investigate the role of CPSF73 in cancer progression?

CPSF73 has been implicated in cancer phenotypes and proposed as a biomarker . To explore this connection:

• Tissue microarray analysis:

  • Use immunohistochemistry with CPSF73 antibodies on cancer tissue arrays

  • Compare expression levels between normal tissues, primary tumors, and metastases

  • Correlate with clinical parameters and patient outcomes

• Chromatin immunoprecipitation (ChIP) approaches:

  • Use ChIP-seq with CPSF73 antibodies to map genome-wide binding sites

  • Identify cancer-specific changes in CPSF73 recruitment to target genes

  • Integrate with RNA-seq data to correlate with altered gene expression

• Functional studies in cancer models:

  • Combine CPSF73 antibodies with small molecule inhibitors of CPSF73

  • Monitor changes in 3' end processing, gene expression, and cancer phenotypes

  • This approach can help validate CPSF73 as a therapeutic target

What are the considerations for studying CPSF73's role in the ubiquitin-proteasome system?

Recent research suggests CPSF73 is regulated post-translationally through ubiquitination :

• Ubiquitination detection approaches:

  • Immunoprecipitate CPSF73 followed by ubiquitin Western blot

  • Use proteasome inhibitors (e.g., MG132) to stabilize ubiquitinated forms

  • Compare ubiquitination patterns in different cellular contexts

• UBE3D interaction studies:

  • Investigate the interaction between CPSF73 and UBE3D (human homolog of yeast Ipa1)

  • Use co-immunoprecipitation with CPSF73 antibodies followed by UBE3D detection

  • Employ UBE3D knockout models to assess effects on CPSF73 stability and function

• Degradation kinetics measurement:

  • Perform cycloheximide chase experiments with CPSF73 antibody detection

  • Compare degradation rates under different cellular conditions

  • This approach can reveal mechanisms regulating CPSF73 protein levels

How can CPSF73 antibodies be utilized in single-cell analysis techniques?

For single-cell level investigations:

• Single-cell Western blot:

  • Adapt traditional Western protocols for microfluidic platforms

  • Optimize CPSF73 antibody concentrations for reduced sample volumes

  • This allows analysis of cell-to-cell variation in CPSF73 expression

• Mass cytometry (CyTOF):

  • Conjugate CPSF73 antibodies with rare earth metals

  • Combine with antibodies against other RNA processing factors

  • This enables high-dimensional analysis of CPSF73 in heterogeneous populations

• Imaging mass cytometry:

  • Apply metal-labeled CPSF73 antibodies to tissue sections

  • Visualize spatial distribution while preserving tissue architecture

  • Correlate with expression of other markers at single-cell resolution

What methodological considerations are important when using CPSF73 antibodies for studying its role in miRNA processing?

CPSF73 is involved in selective processing of microRNAs during cell differentiation :

• RNA immunoprecipitation (RIP) protocol:

  • Cross-link RNA-protein complexes (typically with 1% formaldehyde)

  • Immunoprecipitate with CPSF73 antibodies

  • Analyze associated miRNAs by sequencing or qPCR

  • Compare results between different cellular states (e.g., undifferentiated vs. differentiated)

• In vitro processing assays:

  • Immunodeplete CPSF73 from cell extracts using specific antibodies

  • Assess effects on processing of specific pri-miRNAs (e.g., pri-miR-17-92, pri-miR-290-295)

  • Rescue activity by adding back purified CPSF73 protein

• Live-cell imaging approaches:

  • Generate fluorescently tagged CPSF73 constructs

  • Validate with antibody staining to ensure proper localization

  • Monitor dynamics during miRNA biogenesis in real-time