DIS3L Antibody

What is DIS3L protein and what cellular functions does it perform?

DIS3L (also known as DIS3L1, DIS3-like exonuclease 1) functions as a catalytic component of the RNA exosome complex with 3'->5' exoribonuclease activity. It participates in numerous cellular RNA processing and degradation events . In the cytoplasm, DIS3L is involved in general mRNA turnover and specifically degrades inherently unstable mRNAs containing AU-rich elements (AREs) within their 3' untranslated regions . It also plays a role in RNA surveillance pathways, preventing translation of aberrant mRNAs, and appears to be involved in the degradation of histone mRNA . Unlike its homolog Dis3 which localizes to the nucleus, DIS3L1 is predominantly found in the cytoplasm as confirmed by confocal immunofluorescence microscopy studies .

What are the key structural characteristics of DIS3L protein?

DIS3L is characterized by a molecular weight of approximately 120.8 kilodaltons . The protein contains an RNase II-type RNB domain that is responsible for its exoribonuclease activity . The full-length coding sequence of the largest isoform of human DIS3L1 has been cloned from a teratocarcinoma cDNA library and inserted into various mammalian expression vectors for experimental purposes . Structurally, DIS3L exhibits significant homology with the Dis3 protein family but possesses distinct localization and functional properties that differentiate it from other family members. The protein is widely expressed in human tissues, with notable presence in the brain, liver, and kidney .

What types of DIS3L antibodies are available for research applications?

Multiple types of DIS3L antibodies are available for research applications across various suppliers. These include:

• Rabbit polyclonal antibodies that recognize different epitopes of human DIS3L, such as those targeting recombinant fragments within the N-terminal 1-300 amino acid region .

• Species-specific antibodies with reactivity against human, mouse, and rat DIS3L orthologs .

• Both unconjugated antibodies and conjugated versions (such as APC-conjugated) .

• Antibodies validated for specific applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), and Immunoprecipitation (IP) .

These antibodies vary in their specific applications, recommended dilutions, and validated reactivity with different species, providing researchers with options based on their experimental needs.

How should DIS3L antibodies be optimized for Western blot applications?

For optimal Western blot results with DIS3L antibodies, researchers should consider the following methodology:

• Sample preparation: Use whole cell lysates from appropriate cell lines such as HeLa, HepG2, or K-562 cells, which have been confirmed to express detectable levels of DIS3L protein .

• Protein loading: Load sufficient protein (typically 20-30 μg per lane) to detect the ~121 kDa DIS3L protein.

• Antibody dilution: Starting dilutions of 1:200 to 1:1000 are recommended for most DIS3L antibodies in Western blot applications . Titration may be necessary to determine optimal conditions for each specific antibody.

• Blocking and incubation: Use 5% non-fat milk or BSA in TBST for blocking, and incubate with primary antibody overnight at 4°C for best results.

• Detection: Use appropriate secondary antibodies and sensitive chemiluminescent detection systems to visualize the high molecular weight DIS3L protein.

Researchers should be aware that the observed molecular weight of DIS3L is approximately 121 kDa , which should be considered when interpreting Western blot results.

What are the recommended protocols for immunoprecipitation using DIS3L antibodies?

For successful immunoprecipitation of DIS3L protein, the following methodology is recommended:

• Lysate preparation: Prepare fresh cell lysates using a gentle lysis buffer containing protease inhibitors to preserve protein interactions. K-562 cells have been validated for successful IP of DIS3L .

• Antibody amount: Use 0.5-4.0 μg of DIS3L antibody for immunoprecipitation from 1.0-3.0 mg of total protein lysate .

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Incubation conditions: Incubate lysates with antibody overnight at 4°C under gentle rotation.

• Washing: Perform at least 3-5 stringent washes to remove non-specifically bound proteins.

• Elution and analysis: Elute immunoprecipitated complexes and analyze by Western blot or mass spectrometry.

For co-immunoprecipitation studies investigating DIS3L interactions with exosome complex components, additional considerations for preserving protein-protein interactions are necessary, such as using crosslinking agents or milder washing conditions .

What considerations are important for immunohistochemistry applications with DIS3L antibodies?

When using DIS3L antibodies for immunohistochemistry on paraffin-embedded (IHC-P) tissues, researchers should consider:

• Antigen retrieval: Heat-induced epitope retrieval methods are typically required for formalin-fixed tissues, with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) being common options.

• Blocking: Thorough blocking of endogenous peroxidase activity and non-specific binding sites is essential.

• Antibody selection: Choose antibodies specifically validated for IHC-P applications, as not all DIS3L antibodies perform equally across different applications .

• Controls: Include both positive control tissues known to express DIS3L (such as liver or kidney sections) and negative controls (primary antibody omission).

• Detection system: Use sensitive detection systems appropriate for the expected expression level of DIS3L in target tissues.

• Interpretation: Consider the predominantly cytoplasmic localization pattern of DIS3L when interpreting staining results .

Researchers should be aware that DIS3L shows varying expression levels across different tissue types, with notable presence in brain, liver, and kidney tissues .

How can researchers troubleshoot non-specific binding issues with DIS3L antibodies?

When encountering non-specific binding with DIS3L antibodies, researchers should systematically address potential issues:

• Antibody specificity: Verify the antibody's specificity using positive and negative control samples. Consider using cells with confirmed DIS3L expression (like K-562 or HeLa cells) versus cells where expression has been knocked down via siRNA .

• Blocking optimization: Test different blocking agents (BSA, non-fat milk, normal serum) and concentrations to reduce background.

• Antibody concentration: Titrate the antibody concentration to find the optimal signal-to-noise ratio. For Western blots, starting with higher dilutions (1:500 to 1:1000) may reduce non-specific binding .

• Washing stringency: Increase the number and duration of washes, or adjust detergent concentration in wash buffers.

• Sample preparation: Ensure complete protein denaturation for Western blots, or appropriate fixation for immunohistochemistry.

• Cross-reactivity assessment: If possible, confirm specificity through additional means such as mass spectrometry or using cells from DIS3L knockout models.

For particularly challenging applications, consider using monoclonal antibodies if available, as they generally provide higher specificity than polyclonal antibodies.

What are the common challenges in detecting DIS3L in different subcellular fractions?

Detecting DIS3L in subcellular fractions presents several specific challenges:

• Localization pattern: DIS3L predominantly localizes to the cytoplasm, unlike its homolog Dis3 which is nuclear . This differential localization needs to be considered when fractionating cells.

• Fractionation quality: Ensure clean separation of cytoplasmic and nuclear fractions using markers such as GAPDH (cytoplasmic) and lamin (nuclear) to validate fractionation quality.

• Protein extraction efficiency: High molecular weight proteins like DIS3L (~121 kDa) may require optimized extraction conditions to ensure complete solubilization.

• Compartment-specific controls: Include controls for both cytoplasmic enrichment (e.g., visualizing cytoplasmic markers) and nuclear exclusion when studying DIS3L localization.

• Fixation artifacts: For immunofluorescence applications, different fixation methods can affect the apparent localization of DIS3L. Compare methanol and paraformaldehyde fixation results.

• Resolution limitations: Standard fractionation may not distinguish between different cytoplasmic compartments where DIS3L might preferentially localize.

Confocal microscopy studies have shown that while some nuclear staining may be observed, DIS3L primarily accumulates in the cytoplasm, which should be reflected in fractionation studies .

How can researchers validate the specificity of DIS3L antibodies in their experimental systems?

To validate DIS3L antibody specificity in experimental systems, researchers should implement multiple complementary approaches:

• siRNA knockdown: Transfect cells with DIS3L-specific siRNA and control siRNA, then compare antibody reactivity in Western blots or immunostaining. Successful knockdown should significantly reduce specific signal .

• Overexpression systems: Compare staining patterns in cells overexpressing tagged DIS3L (e.g., GFP-tagged) with antibody staining patterns.

• Peptide competition: Pre-incubate the antibody with its immunogenic peptide before application to samples; specific signals should be diminished.

• Multiple antibodies: Use different antibodies recognizing distinct epitopes of DIS3L and compare results.

• Mass spectrometry: Confirm the identity of the immunoprecipitated protein band by mass spectrometry analysis.

• Cross-species reactivity: If the antibody claims reactivity with multiple species, verify consistent detection of the appropriately sized protein across these species.

• Knockout controls: If available, use DIS3L knockout cell lines or tissues as negative controls.

This multi-faceted validation approach is particularly important for DIS3L, which belongs to a family of related proteins with potential for cross-reactivity .

How can DIS3L antibodies be used to study RNA exosome complex interactions?

DIS3L antibodies can be strategically employed to investigate RNA exosome complex interactions through these methodological approaches:

• Co-immunoprecipitation (Co-IP): Use DIS3L antibodies to immunoprecipitate the protein along with associated exosome components, followed by Western blotting for known exosome proteins such as hRrp40 . This approach has successfully demonstrated that both N- and C-terminally GFP-tagged hDis3L1 proteins co-precipitate with exosome components.

• Reciprocal IP: Immunoprecipitate using antibodies against core exosome components (like hRrp40) and probe for DIS3L to confirm the interaction from both directions .

• Proximity ligation assays: Detect protein-protein interactions in situ using antibodies against DIS3L and other exosome components.

• Size exclusion chromatography: Fractionate cell lysates and probe fractions with DIS3L antibodies to determine which complexes contain DIS3L.

• Functional assays: Compare exoribonuclease activity of immunoprecipitated complexes using DIS3L antibodies versus antibodies against core exosome components, with and without DIS3L knockdown .

These approaches have revealed that hDis3L1 is stably associated with human exosome complexes and contributes to their exoribonuclease activity, unlike human Dis3 which is not detectably associated with the exosome .

What methods can be used to study the role of DIS3L in RNA degradation pathways?

To investigate DIS3L's role in RNA degradation pathways, researchers can implement these methodological approaches:

• RNA degradation assays: Use immunopurified DIS3L (via antibodies) to assess its ability to degrade radiolabeled RNA substrates under various conditions. Studies have shown that GFP-hDis3L1 immunoaffinity purified from transfected cells displays exoribonuclease activity .

• siRNA-mediated knockdown: Deplete DIS3L using specific siRNAs and analyze the accumulation of RNA degradation intermediates. Previous studies showed that knockdown of hDis3L1 resulted in elevated levels of poly(A)-tailed 28S rRNA degradation intermediates .

• RNA immunoprecipitation (RIP): Use DIS3L antibodies to immunoprecipitate the protein along with associated RNA species, followed by RNA sequencing to identify its targets.

• CLIP-seq (Crosslinking and immunoprecipitation followed by sequencing): Identify direct RNA binding targets of DIS3L in vivo.

• Substrate specificity analysis: Compare degradation of different RNA substrates (e.g., structured vs. unstructured, coding vs. non-coding) by immunopurified DIS3L.

• Domain mutation analysis: Express DIS3L with mutations in the RNB domain and compare activity to wild-type protein using antibodies for detection .

Experiments have demonstrated that the exoribonuclease activity associated with hDis3L1 is also associated with the exosome core, and siRNA-mediated depletion of hDis3L1 reduces this activity in anti-hRrp40 precipitates .

How can researchers investigate the differential expression of DIS3L across tissues and disease states?

To investigate differential expression of DIS3L across tissues and disease states, researchers should consider these methodological approaches:

• Tissue microarray analysis: Use DIS3L antibodies validated for IHC to analyze expression patterns across multiple normal and diseased tissues simultaneously.

• Quantitative Western blotting: Compare DIS3L protein levels across tissue lysates or patient samples, normalized to appropriate loading controls.

• Immunofluorescence co-localization: Examine DIS3L localization patterns in relation to other markers in different tissue types or disease states.

• Single-cell analysis: Combine DIS3L antibody staining with single-cell technologies to examine cell-specific expression patterns within heterogeneous tissues.

• Correlation with clinical outcomes: Analyze DIS3L expression levels in patient samples in relation to clinical parameters, disease progression, or treatment response.

• Comparative expression studies: Examine DIS3L expression in relation to other exosome components across tissues or disease models.

While DIS3L is widely expressed with notable presence in tissues such as the brain, liver, and kidney , systematic studies of its differential expression in disease states, particularly in relation to RNA metabolism disorders, would provide valuable insights into its pathophysiological roles.

How might DIS3L antibodies contribute to research on COVID-19 and viral RNA processing?

Recent findings suggest potential roles for DIS3L and the RNA exosome in viral infection contexts, particularly with SARS-CoV-2:

• Exosome component interactions: The search results indicate that "Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication" , suggesting RNA processing machinery including DIS3L may interface with viral replication.

• Methodological approaches: Researchers can use DIS3L antibodies to:

  • Investigate changes in DIS3L localization or expression during viral infection

  • Examine interactions between DIS3L-containing complexes and viral RNA

  • Compare DIS3L activity in infected versus uninfected cells

  • Assess whether DIS3L contributes to viral RNA degradation or stabilization

• Knockdown studies: siRNA-mediated depletion of DIS3L followed by viral challenge can help determine whether this exoribonuclease affects viral replication efficiency.

• Proximity labeling: Using DIS3L antibodies in conjunction with proximity labeling techniques may identify novel interaction partners specifically induced during viral infection.

• Tissue-specific responses: DIS3L antibodies can help investigate how the RNA degradation machinery responds to viral infection in different tissue contexts.

This emerging research area may provide insights into host-pathogen interactions at the level of RNA metabolism and potentially identify new therapeutic targets for viral infections.

What are the current limitations in DIS3L antibody technologies and how might they be addressed?

Current DIS3L antibody technologies face several limitations that affect their research applications:

• Epitope coverage: Most available antibodies target limited regions of the DIS3L protein, potentially missing conformation-specific epitopes that might be functionally relevant. Development of antibodies against diverse epitopes, including conformational epitopes, would expand research capabilities.

• Specificity challenges: Given the homology between DIS3L and related family members, ensuring absolute specificity remains challenging. Advanced antibody validation methods including:

  • Testing in knockout systems

  • Comprehensive cross-reactivity panels

  • Mass spectrometry verification
    would address this limitation.

• Application breadth: While many antibodies work for Western blotting, fewer are validated for immunoprecipitation, ChIP, or super-resolution microscopy. Expanded validation across multiple applications would benefit the research community.

• Species reactivity: Many antibodies are validated only for human samples , limiting comparative studies. Development of antibodies with confirmed cross-species reactivity would enable evolutionary and model organism studies.

• Post-translational modification detection: Current antibodies generally do not distinguish between modified forms of DIS3L. Developing modification-specific antibodies (phospho-specific, etc.) would enable studies of DIS3L regulation.

Addressing these limitations through next-generation antibody development technologies, including recombinant antibodies and synthetic approaches, would significantly advance DIS3L research capabilities.

What are the key considerations for researchers selecting DIS3L antibodies for their experiments?

When selecting DIS3L antibodies for research applications, investigators should consider:

• Application compatibility: Ensure the antibody has been validated for your specific application (Western blot, immunoprecipitation, immunohistochemistry, etc.) as performance can vary significantly between applications .

• Species reactivity: Verify the antibody's reactivity with your experimental model organism. Some DIS3L antibodies are human-specific, while others react with mouse, rat, or other species .

• Epitope information: Consider the antibody's target region within DIS3L. Antibodies targeting different domains may yield different results, particularly in assessing protein interactions or functional studies.

• Validation data: Review available validation data including Western blot images showing the expected 121 kDa band , knockdown/knockout controls, and specificity documentation.

• Experimental conditions: Check recommended dilutions, incubation conditions, and buffer compatibility to ensure optimal performance .

• Source reliability: Select antibodies from established suppliers with good quality control practices and detailed documentation.

• Research objectives: For complex studies examining DIS3L function in RNA degradation, select antibodies validated for maintaining enzymatic activity after immunoprecipitation .

Thorough evaluation of these factors will help researchers select the most appropriate DIS3L antibody for their specific experimental needs, enhancing data quality and reproducibility.