EXOSC3 Antibody

What is EXOSC3 and what role does it play in cellular processes?

EXOSC3 (also known as RRP40, p10, or hRrp-40) is a core component of the RNA exosome complex, which represents a major cellular machinery for processing, surveillance, and turnover of diverse RNA substrates. The RNA exosome complex is essential for viability and highly conserved throughout evolution . EXOSC3 specifically functions as part of the exosome cap, forming a critical structural component that contributes to RNA substrate recognition and processing .

In the nucleus, the RNA exosome complex participates in proper maturation of stable RNA species including ribosomal RNA (rRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA) . EXOSC3 depletion has been shown to destabilize other exosome subunits, including catalytic subunits EXOSC10 and DIS3L, demonstrating its importance for maintaining the structural integrity of the entire complex . Studies using HITS-CLIP (high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation) have revealed that EXOSC3 binds to various RNA targets, with prominent interactions observed with 45S pre-rRNA and 5S rRNA .

EXOSC3 antibodies have been validated for multiple research applications with specific recommended protocols and dilutions. The major applications include:

• Western Blot (WB): Used for detecting EXOSC3 protein in cell and tissue lysates. Typical dilutions range from 1:1000 to 1:4000, with successful detection reported in various cell lines including A2780, HEK-293T, PC-3, and NIH/3T3 cells, as well as mouse spleen tissue .

• Immunoprecipitation (IP): Effective for isolating EXOSC3-containing complexes or associated RNAs. Recommended usage is 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate, with validation in A2780 cells .

• Immunofluorescence/Immunocytochemistry (IF/ICC): Used for cellular localization studies at dilutions of 1:200-1:800, successfully tested in PC-3 cells .

• Flow Cytometry (FACS): Some antibodies like ABIN1498138 have been validated for flow cytometry applications to analyze EXOSC3 expression in cell populations .

• HITS-CLIP: EXOSC3 antibodies have been utilized in high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation to identify RNA targets directly bound to EXOSC3 in vivo .

Each application may require optimization of antibody concentration and experimental conditions, as the performance can vary depending on sample type and preparation methods .

How can I optimize EXOSC3 antibody use for RNA-protein interaction studies?

When investigating RNA-protein interactions involving EXOSC3, researchers commonly employ techniques such as HITS-CLIP or RNA immunoprecipitation (RIP). Based on published methodologies, the following optimization strategies are recommended:

What strategies can be used to validate EXOSC3 antibody specificity?

Validating EXOSC3 antibody specificity is crucial for ensuring reliable experimental results. Several complementary approaches are recommended:

• Knockdown/Knockout Controls: Utilize EXOSC3 knockdown or knockout samples to confirm antibody specificity. Published studies have demonstrated the effectiveness of this approach using inducible shRNAs against EXOSC3 mRNA, which resulted in approximately 70% reduction in EXOSC3 protein levels after 5 days of doxycycline treatment .

• Multiple Antibody Comparison: Use different antibodies targeting distinct epitopes of EXOSC3 to confirm consistent detection patterns. Discrepancies between antibodies may indicate non-specific binding or recognition of different isoforms.

• Molecular Weight Verification: Compare the observed molecular weight with the expected size. EXOSC3 has a calculated molecular weight of 30 kDa and is typically observed at approximately 31 kDa in Western blot applications .

• Peptide Competition Assays: Pre-incubate the antibody with excess immunizing peptide or recombinant EXOSC3 protein before application. Specific binding should be blocked by this competition.

• Mass Spectrometry Validation: For immunoprecipitation experiments, analyze the purified protein complexes by mass spectrometry to confirm the presence of EXOSC3 and known interaction partners.

How does EXOSC3 depletion affect the RNA exosome complex and cellular functions?

EXOSC3 depletion has profound effects on the structure and function of the RNA exosome complex, ultimately impacting various cellular processes:

• Complex Destabilization: EXOSC3 depletion leads to destabilization of other RNA exosome subunits, including the catalytic components EXOSC10 and DIS3L. In experimental systems, maximal EXOSC3 depletion (approximately 70% reduction) correspondingly decreased EXOSC10 levels, indicating structural interdependence among subunits .

• RNA Processing Disruption: The RNA exosome complex normally participates in the processing and maturation of various RNA species. EXOSC3 depletion disrupts these functions, potentially affecting maturation of rRNA, snRNA, and snoRNA .

• RNA Surveillance Impairment: EXOSC3-depleted cells may exhibit defects in RNA quality control mechanisms, potentially leading to accumulation of aberrant RNA species that would normally be degraded by the exosome complex.

• Embryonic Stem Cell Function: Studies in human embryonic stem cells have shown that RNA exosome activity, including EXOSC3 function, plays important roles in regulating stem cell biology, suggesting that EXOSC3 depletion may impact developmental processes .

• Disease Associations: Mutations in EXOSC3 have been linked to neurological disorders, highlighting its importance in normal cellular function, particularly in the nervous system.

When designing experiments involving EXOSC3 depletion, researchers should consider these wide-ranging effects and include appropriate controls to distinguish direct from indirect consequences of EXOSC3 loss.

What are the recommended protocols for Western blotting with EXOSC3 antibodies?

For optimal Western blot results with EXOSC3 antibodies, the following protocol considerations are recommended:

• Sample Preparation:

  • Lyse cells in RIPA buffer or other compatible lysis buffers containing protease inhibitors

  • Sonicate briefly to shear DNA and reduce sample viscosity

  • Centrifuge at 12,000 × g for 20 minutes at 4°C to remove debris

  • Determine protein concentration using Bradford or BCA assay

• Gel Electrophoresis and Transfer:

  • Load 20-50 μg of total protein per lane on 10-12% SDS-PAGE gels

  • Transfer to PVDF or nitrocellulose membranes at 100V for 60-90 minutes or overnight at 30V

• Antibody Incubation:

  • Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary EXOSC3 antibody at recommended dilution (typically 1:1000-1:4000) in blocking buffer overnight at 4°C

  • Wash 3-5 times with TBST, 5 minutes each

  • Incubate with appropriate HRP-conjugated secondary antibody (typically 1:5000-1:10000) for 1 hour at room temperature

  • Wash 3-5 times with TBST, 5 minutes each

• Detection:

  • Apply ECL substrate and expose to X-ray film or image using a digital imaging system

  • Expected band size is approximately 31 kDa, which corresponds to the observed molecular weight of EXOSC3

• Controls and Validation:

  • Include positive control lysates from cells known to express EXOSC3 (e.g., A2780, HEK-293T, PC-3, or NIH/3T3 cells)

  • Consider including EXOSC3-depleted samples as negative controls when available

How should I optimize immunofluorescence protocols using EXOSC3 antibodies?

For successful immunofluorescence staining with EXOSC3 antibodies, consider the following optimization strategies:

• Fixation and Permeabilization:

  • Test both paraformaldehyde (4%, 10-15 minutes) and methanol (-20°C, 10 minutes) fixation methods, as epitope accessibility may differ

  • Permeabilize with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes for paraformaldehyde-fixed cells

  • Methanol fixation typically provides simultaneous permeabilization

• Blocking and Antibody Incubation:

  • Block with 1-5% BSA or normal serum (from the species of the secondary antibody) in PBS for 30-60 minutes

  • Dilute primary EXOSC3 antibody in blocking solution at 1:200-1:800 as recommended

  • Incubate with primary antibody for 1-2 hours at room temperature or overnight at 4°C

  • Wash 3-5 times with PBS, 5 minutes each

  • Incubate with fluorophore-conjugated secondary antibody (typically 1:500-1:1000) for 1 hour at room temperature in the dark

  • Wash 3-5 times with PBS, 5 minutes each

• Counterstaining and Mounting:

  • Counterstain nuclei with DAPI or Hoechst (1:5000) for 5-10 minutes

  • Mount with an anti-fade mounting medium to preserve fluorescence

• Optimization Considerations:

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

  • Test different antigen retrieval methods if necessary

  • Consider using PC-3 cells as positive controls, as these have been validated for EXOSC3 immunofluorescence staining

  • Include negative controls (primary antibody omission and isotype controls)

• Co-localization Studies:

  • For co-localization with other RNA exosome components or RNA processing factors, select antibodies raised in different host species to avoid cross-reactivity

  • Consider sequential staining protocols if antibodies are from the same species

What are the critical parameters for successful immunoprecipitation with EXOSC3 antibodies?

Immunoprecipitation (IP) using EXOSC3 antibodies requires careful optimization of several parameters:

• Lysis Conditions:

  • Use gentle lysis buffers to preserve protein-protein and protein-RNA interactions (e.g., 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA with protease inhibitors)

  • For RNA-IP experiments, include RNase inhibitors in all buffers

  • Adjust salt concentration based on desired stringency (higher salt reduces non-specific binding but may disrupt weak interactions)

• Antibody Amount and Incubation:

  • Use 0.5-4.0 μg of EXOSC3 antibody per 1.0-3.0 mg of total protein lysate as recommended

  • Pre-clear lysate with Protein A/G beads to reduce non-specific binding

  • Incubate lysate with antibody for 2-4 hours or overnight at 4°C with gentle rotation

• Bead Selection and Handling:

  • Choose appropriate beads based on the host species of the EXOSC3 antibody (Protein A for rabbit, Protein G for mouse, or Protein A/G for both)

  • Use 20-50 μl of bead slurry per IP reaction

  • Wash beads 3-5 times with lysis buffer or increasingly stringent wash buffers

  • Elute proteins by boiling in SDS sample buffer or use gentler elution with peptide competition for functional studies

• Controls:

  • Always include an isotype-matched non-immune IgG control processed in parallel

  • For validation, consider using A2780 cells, which have been tested for EXOSC3 immunoprecipitation

• For RNA-Protein Interaction Studies:

  • Consider UV cross-linking prior to lysis (254 nm) as used in HITS-CLIP protocols

  • For stringent RNA target identification, use EXOSC3 antibodies in conjunction with EXOSC4 antibodies to identify RNAs that interact with multiple exosome components

How can I troubleshoot common issues with EXOSC3 antibody applications?

When encountering problems with EXOSC3 antibody experiments, consider the following troubleshooting approaches:

• No Signal or Weak Signal in Western Blot:

  • Verify EXOSC3 expression in your sample; EXOSC3 has been confirmed in A2780, HEK-293T, PC-3, and NIH/3T3 cells

  • Increase protein loading amount (30-50 μg)

  • Reduce antibody dilution (e.g., from 1:4000 to 1:1000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use enhanced sensitivity detection systems

  • Check transfer efficiency with Ponceau S staining

  • Verify that sample preparation maintains protein integrity (use fresh protease inhibitors)

• High Background in Immunofluorescence:

  • Increase blocking time or blocking agent concentration

  • Dilute primary antibody further (e.g., from 1:200 to 1:500)

  • Increase washing steps or duration

  • Use centrifugation or filtration to remove antibody aggregates before use

  • Optimize fixation method (try both methanol and paraformaldehyde)

  • Reduce secondary antibody concentration or switch to highly cross-absorbed versions

• Poor Immunoprecipitation Efficiency:

  • Increase antibody amount within recommended range (0.5-4.0 μg)

  • Extend antibody-lysate incubation time

  • Optimize lysis conditions to better preserve the native conformation of EXOSC3

  • Pre-clear lysate more thoroughly to reduce non-specific binding

  • Verify that the antibody recognizes the native (non-denatured) form of EXOSC3

• Non-specific Bands in Western Blot:

  • Increase blocking time and washing steps

  • Use gradient gels for better separation

  • Verify antibody specificity using EXOSC3 knockdown or knockout controls

  • Consider using alternative EXOSC3 antibodies targeting different epitopes for confirmation

What are the recommended quality control measures for EXOSC3 antibody experiments?

To ensure reproducible and reliable results with EXOSC3 antibodies, implement the following quality control measures:

• Antibody Validation:

  • Verify antibody specificity using EXOSC3 knockdown/knockout samples

  • Confirm expected molecular weight (30-31 kDa) in Western blot applications

  • Cross-validate results with multiple antibodies targeting different EXOSC3 epitopes

  • Document antibody lot number, as performance can vary between lots

• Positive and Negative Controls:

  • Include positive control samples with known EXOSC3 expression (e.g., A2780, HEK-293T, PC-3, or NIH/3T3 cells)

  • Use appropriate negative controls (isotype-matched IgG for IP, primary antibody omission for IF)

  • For IP-based RNA studies, include RNase treatment controls to distinguish direct protein binding from RNA-mediated interactions

• Quantification and Reproducibility:

  • Perform experiments in triplicate to ensure reproducibility

  • Use appropriate quantification methods (densitometry for Western blots, fluorescence intensity for IF)

  • Report statistical analyses for quantitative comparisons

  • Document detailed experimental conditions to enable reproducibility

• Cross-Validation with Alternative Methods:

  • Confirm key findings using orthogonal approaches (e.g., validate Western blot results with IF)

  • For protein-protein interaction studies, confirm IP results with alternative techniques like proximity ligation assay

  • For RNA binding studies, validate HITS-CLIP results with RIP-qPCR for selected targets

• Documentation and Reporting:

  • Maintain detailed records of antibody source, catalog number, lot number, and dilutions used

  • Document all experimental conditions, including buffers, incubation times, and temperatures

  • Report all controls and validation experiments in publications and presentations

What are the emerging applications and future directions for EXOSC3 antibody research?

As our understanding of RNA biology continues to evolve, EXOSC3 antibodies are becoming increasingly valuable tools for investigating RNA processing mechanisms. Several emerging applications and future directions deserve consideration:

• Single-Cell Analysis: Adapting EXOSC3 antibodies for single-cell proteomics and RNA-protein interaction studies will provide insights into cell-to-cell variability in RNA processing mechanisms.

• Super-Resolution Microscopy: Utilizing EXOSC3 antibodies with super-resolution microscopy techniques will enhance our understanding of the spatial organization of RNA exosome complexes within cellular compartments.

• Therapeutic Target Validation: Given the association between EXOSC3 mutations and certain neurological disorders, antibodies will play crucial roles in validating therapeutic approaches targeting the RNA exosome complex.

• Development of Proximity-Based Assays: Adapting EXOSC3 antibodies for proximity ligation assays (PLA) or BioID approaches will provide comprehensive maps of protein-protein interactions involving the RNA exosome.

• Combinatorial Omics Approaches: Integrating EXOSC3 antibody-based techniques (such as HITS-CLIP) with transcriptomics, proteomics, and genomics data will provide holistic views of RNA exosome functions across different cellular contexts.