CPSF100 Antibody

What is CPSF100 and what is its functional significance in cellular processes?

CPSF100 (also known as CPSF2) is a key subunit of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex that plays a critical role in pre-mRNA 3'-end formation. This protein works by recognizing the AAUAAA signal sequence and interacting with poly(A) polymerase and other factors to facilitate cleavage and poly(A) addition to pre-mRNAs . The proper functioning of CPSF100 is essential for accurate processing of mRNA 3' ends, which affects mRNA stability, nuclear export, and translation efficiency. In Arabidopsis, CPSF100 (AtCPSF100) is part of a five-protein CPSF complex that also includes CPSF160, CPSF73-I, CPSF73-II, and CPSF30 . In humans, CPSF100 is additionally involved in histone 3' end pre-mRNA processing, highlighting its broader role in RNA metabolism beyond just polyadenylated mRNAs .

What types of CPSF100 antibodies are available for research applications?

Research laboratories can access several types of CPSF100 antibodies, each with specific characteristics suitable for different experimental approaches:

• Polyclonal antibodies: Products like ab229114 are rabbit polyclonal antibodies that offer high sensitivity and are suitable for Western blot (WB) and immunocytochemistry/immunofluorescence (ICC/IF) applications . Polyclonal antibodies recognize multiple epitopes on the CPSF100 protein, potentially providing stronger signals.

• Monoclonal antibodies: Products like ab126760 (clone EPR7599) are rabbit recombinant monoclonal antibodies that provide high specificity and reproducibility for Western blot applications . These antibodies recognize specific epitopes and typically offer reduced background compared to polyclonal alternatives.

• Species-specific antibodies: Some antibodies are designed for plant research (e.g., Arabidopsis thaliana CPSF100), while others target mammalian systems (human, mouse, rat) . The choice depends on the experimental model organism.

What are the recommended experimental applications for CPSF100 antibodies?

Based on available validation data, CPSF100 antibodies are suitable for multiple experimental applications:

When designing experiments, researchers should verify that the antibody has been validated for their specific application and species of interest. For example, ab126760 has been specifically tested and validated for Western blot applications with human, mouse, and rat samples , while other antibodies might have broader or more limited species reactivity.

How can researchers optimize Western blot protocols for CPSF100 detection?

Optimizing Western blot protocols for CPSF100 detection requires careful consideration of several parameters:

• Sample preparation: For optimal CPSF100 detection, prepare whole cell lysates using denaturing conditions. Based on published protocols, cells should be lysed in buffer containing SDS and protease inhibitors to prevent degradation of the target protein.

• Gel selection: Use 7.5% SDS-PAGE gels for optimal separation of CPSF100, which has a predicted molecular weight of 88 kDa . This percentage allows for better resolution of proteins in this size range.

• Antibody conditions:

  • Primary antibody: Use anti-CPSF100 antibodies at a dilution of 1:1000 (as validated for ab229114 and ab126760)

  • Secondary antibody: For rabbit primary antibodies, use HRP-conjugated goat anti-rabbit at 1:2000 dilution

  • Incubation: Overnight at 4°C for primary antibody followed by 1-2 hours at room temperature for secondary antibody

• Loading control: Include appropriate loading controls based on subcellular localization, as CPSF100 is predominantly nuclear.

• Blocking and washing: Use 5% non-fat milk or BSA in TBST for blocking, and perform stringent washing steps (4-5 times with TBST) to minimize background.

What approaches can be used to validate the specificity of CPSF100 antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For CPSF100 antibodies, consider these validation approaches:

• Genetic validation: Use CPSF100 knockout or knockdown (siRNA/shRNA) cells/tissues as negative controls. The absence or reduction of signal at the expected molecular weight of 88 kDa would confirm specificity .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples. Specific binding should be blocked by the peptide, resulting in loss of signal.

• Cross-reactivity assessment: Test the antibody against samples from multiple species to confirm predicted cross-reactivity. For example, PHY0840A has been reported to cross-react with multiple plant species including Arabidopsis thaliana, Brassica species, and various crop plants .

• Multiple antibody comparison: Use different antibodies targeting distinct epitopes of CPSF100 and compare detection patterns. Consistent patterns would support specificity.

• Mass spectrometry verification: Perform immunoprecipitation followed by mass spectrometry to confirm that the antibody is pulling down CPSF100 and expected interacting partners like other CPSF complex components.

How should researchers interpret contradictory results when studying CPSF100 using different antibodies?

When facing contradictory results with different CPSF100 antibodies, consider these systematic approaches:

• Epitope mapping analysis: Different antibodies may target different epitopes on CPSF100, which could be differentially accessible depending on protein conformation, post-translational modifications, or protein-protein interactions. Map the epitopes recognized by each antibody to understand potential differences.

• Isoform specificity: Verify whether the contradictory results might stem from detection of different CPSF100 isoforms. Some antibodies might recognize specific splice variants or post-translationally modified forms.

• Technical validation:

  • Confirm that each antibody was used under its optimal conditions

  • Verify buffer compatibility and storage conditions were maintained

  • Check for batch-to-batch variations in antibody production

• Biological context considerations: Evaluate whether differences are due to biological variation (cell type, developmental stage, species differences) rather than antibody performance issues.

• Independent verification: Use complementary approaches like mass spectrometry or RNA-based methods (RT-PCR, RNA-seq) to independently verify CPSF100 expression patterns or functions being studied.

What are the critical controls needed when using CPSF100 antibodies in research?

Proper experimental controls are essential for generating reliable and interpretable data with CPSF100 antibodies:

• Positive controls:

  • Cell lines with verified CPSF100 expression: HeLa, Ramos, Jurkat, and 293T cells have been validated for CPSF100 expression and can serve as positive controls for human studies

  • Recombinant CPSF100 protein: Can be used as a standard for size verification and antibody validation

• Negative controls:

  • Primary antibody omission: Samples processed without primary antibody to assess secondary antibody non-specific binding

  • Isotype controls: Samples incubated with non-specific antibodies of the same isotype

  • CPSF100-depleted samples: Cells treated with CPSF100-targeting siRNA/shRNA

• Specificity controls:

  • Peptide competition assays: Pre-incubation of antibody with immunizing antigen

  • Multiple antibody verification: Using different antibodies targeting different CPSF100 epitopes

• Technical controls:

  • Loading controls appropriate for the subcellular fraction being analyzed

  • Sample processing controls to ensure equal treatment across experimental conditions

How can researchers effectively study CPSF100 interactions with other members of the CPSF complex?

Studying CPSF100 interactions with other CPSF complex components requires carefully designed approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-CPSF100 antibodies to pull down CPSF100 and associated proteins

  • Probe for other CPSF components (CPSF160, CPSF73, CPSF30) in Western blots of immunoprecipitated material

  • Consider crosslinking approaches to capture transient interactions

• Reciprocal Co-IP:

  • Perform reverse Co-IP using antibodies against other CPSF components to confirm interactions

  • Compare results across multiple immunoprecipitation approaches to build confidence in interaction data

• Proximity ligation assays (PLA):

  • Use pairs of antibodies against CPSF100 and other CPSF components

  • PLA signals indicate proximity (<40 nm) between proteins, supporting in situ interaction

• Sucrose gradient fractionation:

  • Separate cellular components by density

  • Analyze fractions by Western blot using CPSF100 antibodies

  • Co-fractionation of CPSF components supports complex formation

• Functional validation:

  • Design assays to assess how disruption of CPSF100 affects function of other CPSF components

  • Monitor polyadenylation activity when CPSF100 is depleted or mutated

What is the optimal protocol for immunofluorescence detection of CPSF100?

For optimal immunofluorescence detection of CPSF100, follow this methodological approach:

• Cell preparation and fixation:

  • Grow cells on coverslips to 70-80% confluence

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes

• Blocking and antibody incubation:

  • Block with 5% normal serum in PBS for 1 hour at room temperature

  • Incubate with primary anti-CPSF100 antibody (such as ab229114) at 1:100-1:250 dilution overnight at 4°C

  • Wash 3x with PBS

  • Incubate with fluorophore-conjugated secondary antibody at 1:500 dilution for 1 hour at room temperature

  • Wash 3x with PBS

• Nuclear counterstaining and mounting:

  • Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes

  • Mount slides with anti-fade mounting medium

• Imaging considerations:

  • CPSF100 typically shows nuclear localization with potential enrichment in nuclear speckles

  • Use confocal microscopy for optimal resolution of nuclear structures

  • Consider co-staining with markers of nuclear speckles (SC35) or other RNA processing factors

• Controls and validation:

  • Include primary antibody omission controls

  • Consider CPSF100 knockdown cells as negative controls

  • Co-stain with other CPSF complex components to confirm co-localization

How should researchers prepare samples for optimal CPSF100 detection in different cellular contexts?

Sample preparation strategies should be tailored to the specific cellular context and research question:

• Whole cell lysates for Western blotting:

  • Lyse cells in RIPA buffer supplemented with protease inhibitors

  • Sonicate briefly to shear DNA and reduce viscosity

  • Centrifuge at 14,000 × g for 10 minutes to remove debris

  • Quantify protein concentration and load 10-30 μg per lane

• Nuclear extracts for studying CPSF100 in its functional context:

  • Isolate nuclei using hypotonic buffer and detergent-based methods

  • Extract nuclear proteins with high-salt buffer (300-400 mM NaCl)

  • This approach enriches for nuclear CPSF100 and enhances detection sensitivity

• Tissue samples:

  • Fresh tissues: Homogenize in appropriate buffer with protease inhibitors

  • Fixed tissues: For immunohistochemistry, use antigen retrieval methods (citrate buffer, pH 6.0 at 95°C for 20 minutes) before antibody incubation

  • Consider tissue-specific optimization, as CPSF100 detection may vary between tissue types

• Plant samples (for AtCPSF100):

  • Use specialized plant protein extraction buffers containing PVPP to remove phenolic compounds

  • Optimize extraction for different plant tissues (leaves, roots, flowers)

  • Consider developmental stage effects on AtCPSF100 expression

How can CPSF100 antibodies be utilized to study RNA processing defects in disease models?

CPSF100 antibodies can provide valuable insights into RNA processing defects in various disease contexts:

• Cancer research applications:

  • Compare CPSF100 expression and localization between normal and malignant cells

  • Investigate correlation between CPSF100 alteration and alternative polyadenylation patterns in tumors

  • Assess CPSF100 complex integrity in cancer cells with altered mRNA processing

• Neurodegenerative disease studies:

  • Examine CPSF100 localization in models of neurodegeneration

  • Investigate potential sequestration of CPSF100 in protein aggregates

  • Correlate CPSF100 function with altered RNA processing observed in diseases like ALS or Alzheimer's

• Methodological approaches:

  • Immunohistochemistry of patient samples using validated CPSF100 antibodies

  • Co-localization studies with disease-associated proteins

  • Sequential extraction protocols to examine CPSF100 in soluble vs. insoluble fractions

  • Chromatin immunoprecipitation to assess CPSF100 recruitment to target genes

• Functional validation experiments:

  • Rescue experiments in disease models by modulating CPSF100 levels

  • Analysis of polyadenylation site usage following CPSF100 perturbation

  • Correlation of CPSF100 function with disease phenotypes

What are the considerations for using CPSF100 antibodies in chromatin immunoprecipitation (ChIP) experiments?

While CPSF100 antibodies have not been specifically validated for ChIP in the provided search results, researchers considering this application should consider:

• Antibody selection criteria:

  • Choose antibodies raised against native protein rather than denatured epitopes

  • Monoclonal antibodies may offer higher specificity for ChIP applications

  • Test multiple antibodies as ChIP efficiency can vary significantly between different antibodies to the same protein

• Crosslinking optimization:

  • CPSF100 interacts with chromatin indirectly through RNA and other proteins

  • Test both formaldehyde (protein-protein/protein-DNA crosslinks) and UV (protein-RNA crosslinks) approaches

  • Consider dual crosslinking protocols for optimal capture of CPSF100 at chromatin

• Control considerations:

  • Input controls are essential for normalization

  • IgG controls assess non-specific binding

  • Positive controls should target regions known to undergo polyadenylation

  • Negative controls should examine regions without polyadenylation sites

• Data analysis approach:

  • CPSF100 likely shows enrichment near transcription termination sites

  • Compare CPSF100 binding with RNA Pol II occupancy and other 3' processing factors

  • Correlate with transcription termination and polyadenylation efficiency

How should researchers troubleshoot non-specific bands when using CPSF100 antibodies in Western blot?

When encountering non-specific bands with CPSF100 antibodies, implement this systematic troubleshooting approach:

• Confirm target band identification:

  • The expected molecular weight for human CPSF100 is approximately 88 kDa

  • Verify using positive control samples (HeLa, Ramos, Jurkat, or 293T cell lysates)

• Optimize blocking conditions:

  • Test different blocking agents (5% milk, 5% BSA, commercial blocking buffers)

  • Increase blocking time (2-3 hours at room temperature or overnight at 4°C)

• Adjust antibody conditions:

  • Titrate primary antibody concentration (try higher dilutions: 1:2000 or 1:5000)

  • Reduce incubation time or temperature

  • Add 0.1-0.5% Tween-20 to antibody diluent to reduce non-specific binding

• Improve washing protocol:

  • Increase number of washes (5-6 times for 10 minutes each)

  • Use higher Tween-20 concentration in wash buffer (0.1-0.2%)

• Sample preparation modifications:

  • Ensure complete protein denaturation (boil samples for 5-10 minutes)

  • Add protein phosphatase inhibitors to preserve post-translational modifications

  • Filter lysates to remove particulates that might cause artifact bands

• Validation approaches:

  • Run peptide competition assays to identify which bands are specific

  • Compare patterns across multiple CPSF100 antibodies

  • Test CPSF100-depleted samples to confirm which bands disappear

What approaches can help distinguish between CPSF100 and related proteins in experimental systems?

Distinguishing CPSF100 from related proteins requires careful experimental design:

• Molecular characteristics for differentiation:

  • CPSF100 has a predicted molecular weight of 88 kDa in humans

  • In Arabidopsis, AtCPSF100 can be distinguished from other CPSF components (CPSF160, CPSF73-I, CPSF73-II, and CPSF30) by size

• Methodological approaches:

  • Use higher resolution gels (8-10%) for better separation of CPSF components

  • Perform 2D gel electrophoresis to separate based on both size and isoelectric point

  • Employ isoform-specific antibodies when available

• RNA interference validation:

  • Design specific siRNAs targeting CPSF100 but not related proteins

  • Confirm which bands/signals are reduced following CPSF100 knockdown

• Mass spectrometry confirmation:

  • Excise bands from gels for protein identification by mass spectrometry

  • Compare peptide sequences to confirm protein identity

• Co-immunoprecipitation patterns:

  • Different CPSF components will co-precipitate with distinct sets of interacting proteins

  • Compare immunoprecipitation profiles to distinguish CPSF100 from related proteins

How might emerging technologies enhance the application of CPSF100 antibodies in research?

Emerging technologies offer new opportunities for CPSF100 research:

• Proximity labeling approaches:

  • BioID or APEX2 fusion to CPSF100 to identify proximal proteins in living cells

  • Helps map the dynamic CPSF100 interactome under different conditions

• Super-resolution microscopy:

  • STORM or PALM imaging with CPSF100 antibodies

  • Reveals nanoscale organization of 3' processing machinery within the nucleus

• Single-molecule imaging:

  • Track individual CPSF100 molecules in living cells

  • Provides insights into dynamics and residence time at sites of RNA processing

• CRISPR-based approaches:

  • Endogenous tagging of CPSF100 for live-cell imaging

  • Creation of conditional knockout models for functional studies

• Antibody engineering advances:

  • Development of recombinant nanobodies against CPSF100

  • Creation of bispecific antibodies targeting CPSF100 and other complex components

What are the considerations for studying CPSF100 function in different model organisms?

When extending CPSF100 research across diverse model organisms, consider:

• Cross-reactivity verification:

  • Confirm antibody reactivity with the target species before proceeding

  • The PHY0840A antibody has been reported to cross-react with multiple plant species including Arabidopsis thaliana, Brassica species, and various crop plants

  • Mammalian CPSF100 antibodies typically work with human, mouse, and rat samples

• Evolutionary conservation analysis:

  • CPSF100 is highly conserved, but species-specific variations exist

  • When using antibodies across species, verify epitope conservation in sequence alignments

• Species-specific experimental considerations:

  • Plant systems: Consider tissue-specific extraction methods for different plant tissues

  • Invertebrate models: Optimize fixation protocols for immunohistochemistry

  • Vertebrate models: Account for tissue-specific expression patterns

• Functional conservation testing:

  • Verify whether CPSF100 function in polyadenylation is conserved across species

  • Test complementation of CPSF100 mutants across species boundaries

• Species-appropriate controls:

  • Use species-matched positive controls when available

  • Consider generating species-specific validation tools for novel model organisms

By addressing these considerations, researchers can effectively extend CPSF100 studies across evolutionary boundaries while maintaining experimental rigor.