DIS3L2 Antibody

What is DIS3L2 and why is it significant in research?

DIS3L2 (DIS3-like exonuclease 2) is a highly conserved 3'-5' exoribonuclease that plays crucial roles in RNA metabolism and degradation pathways. It has gained significant research interest because mutations in the DIS3L2 gene are associated with Perlman syndrome and Wilms' tumor of the kidney, both human overgrowth disorders . The protein is particularly important for controlling cell proliferation and tissue growth through the degradation of specific RNA targets. Recent research has demonstrated that DIS3L2 specifically targets uridylated RNAs for degradation, including pre-let-7 miRNA precursors, which are critical regulators of development and differentiation . The study of DIS3L2 provides insights into fundamental RNA degradation mechanisms and their connection to human disease.

What types of DIS3L2 antibodies are available for research applications?

Several types of DIS3L2 antibodies are available for research applications, including:

When selecting an antibody, researchers should consider their specific application requirements, target species, and whether they need to detect specific isoforms or post-translationally modified forms of DIS3L2 .

What are the optimal conditions for using DIS3L2 antibodies in Western blot applications?

For Western blot applications with DIS3L2 antibodies, researchers should follow these methodological guidelines:

• Sample preparation: Prepare cell or tissue lysates using standard protocols with protease inhibitors to prevent protein degradation.

• Loading amount: Based on available data, antibodies perform optimally at dilutions of 1:200-1:2000 for polyclonal rabbit antibodies and 0.04-0.4 μg/mL for affinity-isolated antibodies .

• Gel separation: Use 8-10% SDS-PAGE gels to achieve good separation around the 65-99 kDa range where DIS3L2 is typically detected.

• Expected bands: Prepare to visualize two potential bands at approximately 65 kDa and 99 kDa, corresponding to potential isoforms or processed forms of DIS3L2 .

• Controls: Include positive controls such as HepG2 cell lysates for human samples or mouse embryo tissue for mouse samples, which have been validated with commercially available antibodies.

• Blocking: Use 5% non-fat milk or BSA in TBST for blocking, depending on the specific antibody manufacturer's recommendations.

Researchers should be aware that the observed molecular weights (65 kDa and 99 kDa) may differ from the calculated molecular weight, which could be due to post-translational modifications or proteolytic processing of the protein .

How can DIS3L2 antibodies be used to investigate its subcellular localization?

Investigating DIS3L2 subcellular localization is crucial for understanding its function, as it has been shown to be predominantly cytoplasmic, unlike other DIS3 family members . To effectively study DIS3L2 localization:

• Immunofluorescence approach:

  • Fix cells using 4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilize with 0.1-0.5% Triton X-100

  • Block with 1-5% BSA or normal serum

  • Incubate with DIS3L2 primary antibody (typical dilutions 1:200-1:500 for immunohistochemistry applications)

  • Use appropriate fluorophore-conjugated secondary antibodies

  • Co-stain with markers for subcellular compartments (e.g., DAPI for nucleus, ER markers)

• Subcellular fractionation approach:

  • Separate cytoplasmic and nuclear fractions using established fractionation protocols

  • Perform Western blot analysis on the separate fractions

  • Probe with DIS3L2 antibody and compartment-specific markers as controls

• Data interpretation:

  • DIS3L2 is expected to show predominantly cytoplasmic localization

  • Any nuclear localization or change in localization under specific conditions should be carefully validated with multiple techniques

This combined approach will provide robust data on DIS3L2 localization in various cell types or experimental conditions.

What are the recommended approaches for studying DIS3L2-RNA interactions using immunoprecipitation?

RNA immunoprecipitation (RIP) and related techniques are valuable for studying DIS3L2-RNA interactions, particularly since DIS3L2 has been shown to target specific RNAs like uridylated pre-let-7 . A comprehensive approach includes:

• Standard RIP protocol:

  • Cross-link RNA-protein complexes in vivo using formaldehyde (optional but recommended for transient interactions)

  • Lyse cells in non-denaturing conditions with RNase inhibitors

  • Pre-clear lysates with protein A/G beads

  • Immunoprecipitate with DIS3L2 antibody (or mock IgG control)

  • Isolate RNA from immunoprecipitates

  • Analyze by RT-qPCR or RNA sequencing

• Enhanced detection of transient interactions:

  • Consider using catalytically inactive DIS3L2 (D391N mutant) as demonstrated in previous studies, which enhanced detection of uridylated pre-let-7 due to reduced turnover of bound substrates

  • This approach revealed that the D391N mutant effectively co-precipitated with uridylated pre-let-7 miRNA, while wild-type DIS3L2 showed less association due to rapid substrate degradation

• Analysis of oligo(U)-tailed RNA targets:

  • Include specialized techniques for 3' end analysis such as 3' RACE or circularization RT-PCR

  • Compare results with published CLIP-seq data on DIS3L2-targeted RNAs

• Controls and validation:

  • Include known DIS3L2 targets such as uridylated pre-let-7 as positive controls

  • Use non-target RNAs as negative controls

  • Validate with reciprocal approaches such as RNA pull-down with biotinylated RNA

These methodologies will provide comprehensive insights into the specific RNAs targeted by DIS3L2 in your experimental system.

How should researchers address the detection of multiple DIS3L2 bands in Western blot analysis?

The detection of multiple DIS3L2 bands (commonly at 65 kDa and 99 kDa) in Western blot analysis requires careful interpretation and validation:

• Validation of band specificity:

  • Perform peptide competition assays where the antibody is pre-incubated with the immunizing peptide

  • Use siRNA or CRISPR knockout of DIS3L2 to confirm which bands are specific

  • Compare results with multiple DIS3L2 antibodies targeting different epitopes

• Investigation of potential causes:

  • Alternative splicing: Analyze RNA-seq data or perform RT-PCR to detect alternative transcripts

  • Post-translational modifications: Use phosphatase treatment to check for phosphorylation

  • Proteolytic processing: Include protease inhibitors during sample preparation and compare fresh vs. stored samples

  • Different isoforms: DIS3L2 has annotated isoforms of different lengths

• Technical optimization:

  • Adjust SDS-PAGE conditions (gel percentage, running time)

  • Optimize transfer conditions for high molecular weight proteins

  • Test different blocking reagents (milk vs. BSA)

  • Adjust antibody concentration and incubation time

• Cell/tissue specificity:

  • Compare DIS3L2 expression patterns across different cell types or tissues

  • Document any cell-type specific band patterns for future reference

This systematic approach will help distinguish between technical artifacts and biologically relevant DIS3L2 variants.

What are the key considerations for optimizing immunohistochemical detection of DIS3L2?

Optimizing immunohistochemical (IHC) detection of DIS3L2 requires attention to several critical factors:

• Fixation and antigen retrieval:

  • Test multiple fixatives (formalin, paraformaldehyde, methanol)

  • Optimize antigen retrieval methods (heat-induced in citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Determine optimal retrieval time (10-30 minutes)

• Antibody selection and validation:

  • Use antibodies specifically validated for IHC applications, such as the affinity-isolated antibody described in search result

  • Validate antibody specificity using tissues from DIS3L2 knockout models or with siRNA knockdown controls when possible

  • Test recommended dilution ranges (1:200-1:500 for the antibody in search result )

• Detection systems:

  • Compare polymer-based vs. avidin-biotin detection systems

  • Consider amplification methods for low-abundance proteins

  • Test chromogens for optimal signal-to-noise ratio

• Controls:

  • Include positive control tissues with known DIS3L2 expression

  • Use appropriate negative controls (omission of primary antibody, isotype controls)

  • Include tissues from DIS3L2 knockout or knockdown models when available

• Interpretation guidelines:

  • DIS3L2 is predominantly cytoplasmic , so primarily cytoplasmic staining should be expected

  • Document and investigate any unexpected nuclear or organelle-specific staining

  • Quantify staining intensity and distribution using appropriate digital image analysis if needed

Following these guidelines will help ensure specific and reproducible detection of DIS3L2 in tissue sections.

How can researchers validate the specificity of DIS3L2 antibodies for their experimental system?

Validating antibody specificity is crucial for reliable DIS3L2 research. A comprehensive validation strategy includes:

• Genetic approaches:

  • Generate CRISPR-Cas9 knockout models as described in previous DIS3L2 studies

  • Use siRNA or shRNA knockdown of DIS3L2

  • Compare antibody signals between control and DIS3L2-depleted samples across multiple applications (WB, IHC, IF)

• Expression system validation:

  • Overexpress tagged DIS3L2 (FLAG, HA, or GFP-tagged) and confirm co-detection with the DIS3L2 antibody

  • Use both wild-type DIS3L2 and the catalytically inactive D391N mutant

  • Test detection of different known isoforms of DIS3L2

• Cross-reactivity assessment:

  • Test the antibody against related family members (DIS3, DIS3L1)

  • Perform epitope mapping to confirm target specificity

  • Check for species cross-reactivity if working with non-human models

• Functional validation:

  • Confirm that the antibody can immunoprecipitate active DIS3L2 by testing the exonuclease activity of immunoprecipitates

  • Verify the antibody's ability to detect phenotypic changes associated with DIS3L2 manipulation, such as effects on tissue growth or ER-targeted translation

• Reproducibility testing:

  • Compare results across different lots of the same antibody

  • Compare results from different antibodies targeting different epitopes of DIS3L2

This comprehensive validation approach ensures reliable detection of DIS3L2 and increases confidence in experimental results.

How can DIS3L2 antibodies be utilized to investigate its role in ER-targeted mRNA translation?

Recent research has uncovered a critical role for DIS3L2 in ER-targeted mRNA translation . To investigate this function:

• Co-localization studies:

  • Perform double immunofluorescence with DIS3L2 antibodies and ER markers

  • Use super-resolution microscopy to precisely define spatial relationships

  • Analyze changes in localization under ER stress conditions

• Functional translation assays:

  • Implement secreted luciferase reporter assays as described in previous studies

  • Compare translation efficiency of ER-targeted versus cytosolic proteins in DIS3L2 knockout or knockdown models

  • Analyze polysome profiles specific to ER-associated ribosomes

• Molecular mechanism investigation:

  • Use DIS3L2 antibodies for RNA immunoprecipitation followed by sequencing (RIP-seq) to identify associated ER-targeted mRNAs

  • Perform pulse-chase labeling experiments to measure the impact of DIS3L2 depletion on the synthesis and secretion rates of specific proteins

  • Investigate interactions between DIS3L2 and components of the signal recognition particle (SRP) or SRP receptor

• 7SL RNA surveillance:

  • Examine the relationship between DIS3L2 and 7SL RNA (component of SRP) as indicated in previous studies

  • Compare levels of truncated versus full-length 7SL RNA in control versus DIS3L2-depleted cells

  • Test rescue of ER translation defects by overexpression of wild-type 7SL RNA

These approaches will provide mechanistic insights into how DIS3L2 contributes to the surveillance of 7SL RNA and regulation of ER-targeted translation.

What methodologies can be used to investigate the interplay between DIS3L2 and the RNA uridylation pathway?

DIS3L2 has been identified as an oligo(U)-binding exonuclease that specifically targets uridylated RNAs . To study this interplay:

• Terminal uridyltransferase (TUTase) interaction studies:

  • Perform co-immunoprecipitation assays using DIS3L2 antibodies to detect interactions with TUT4/7

  • Conduct proximity ligation assays to visualize potential interactions in situ

  • Analyze how these interactions change under various cellular conditions

• Uridylated RNA substrate analysis:

  • Implement TAIL-seq or similar methodologies to globally profile RNA 3' ends and uridylation status

  • Compare uridylated RNA profiles between wild-type and DIS3L2-depleted cells

  • Focus on known substrates like pre-let-7 miRNA as well as novel targets

• Mechanistic experiments:

  • Test how TUT4/7 knockdown affects DIS3L2 function and substrate targeting

  • As demonstrated in previous research, examine whether TUT4/7 knockdown can rescue phenotypes in DIS3L2 knockout cells

  • Analyze the structural requirements for DIS3L2 recognition of uridylated substrates

• Functional consequence assessment:

  • Measure the impact on miRNA expression profiles, particularly let-7 family members

  • Examine downstream effects on proliferation and differentiation

  • Assess consequences for ER-targeted translation as demonstrated in previous studies

This integrated approach will provide comprehensive insights into the functional relationship between the uridylation machinery and DIS3L2-mediated RNA degradation.

How can researchers investigate the role of DIS3L2 in cellular growth control and cancer-related pathways?

Given the association of DIS3L2 mutations with Perlman syndrome and Wilms' tumor , investigating its role in growth control is of high clinical relevance:

• Cell proliferation and growth assays:

  • Compare proliferation rates between DIS3L2 wild-type, knockdown, and knockout cells using methodologies described in previous studies

  • Analyze cell cycle profiles using flow cytometry

  • Measure changes in tissue/organ size in appropriate model systems (as observed in Drosophila wing imaginal disc studies)

• Genetic interaction studies:

  • Perform genetic rescue experiments using wild-type versus catalytically inactive DIS3L2 mutants

  • Test for synthetic lethality or growth defects when combining DIS3L2 deficiency with mutations in other growth control genes

  • Implement CRISPR screens to identify genetic modifiers of DIS3L2-associated phenotypes

• RNA target identification in growth control:

  • Use DIS3L2 antibodies for RNA immunoprecipitation followed by high-throughput sequencing

  • Compare RNA profiles between normal tissues and Wilms' tumors or other DIS3L2-associated malignancies

  • Focus analysis on non-coding RNAs (tRNAs, snRNAs, snoRNAs) which have been identified as major DIS3L2 targets

• Signaling pathway analysis:

  • Investigate the relationship between DIS3L2 and established growth control pathways

  • Previous research has shown that DIS3L2 depletion results in enhanced proliferation through well-known cellular pathways

  • Use phospho-specific antibodies to monitor activation status of relevant signaling molecules

These methodologies will provide insights into how DIS3L2 functions as a tumor suppressor and regulator of tissue growth.

What are the emerging applications of DIS3L2 antibodies in clinical research?

The connection between DIS3L2 mutations and human overgrowth disorders opens several promising clinical research applications:

• Diagnostic biomarker development:

  • Investigate DIS3L2 protein expression patterns in normal versus pathological tissues

  • Evaluate whether antibody-based detection of DIS3L2 levels or localization could serve as a biomarker for certain cancer types

  • Study correlations between DIS3L2 expression and clinical outcomes in relevant cancers

• Therapeutic targeting approaches:

  • Use antibodies to identify interacting partners that might represent druggable targets

  • Develop screening assays for compounds that could modulate DIS3L2 activity

  • Explore whether restoring normal DIS3L2 function could slow growth in relevant cancer models

• Personalized medicine applications:

  • Determine if DIS3L2 expression levels predict response to certain therapies

  • Study whether DIS3L2 mutations affect drug sensitivity profiles

  • Develop patient-derived models to test targeted approaches

These emerging applications highlight the translational potential of DIS3L2 research beyond basic mechanisms, particularly in overgrowth syndromes and related cancers.

How might researchers address contradictory data regarding DIS3L2 function across different experimental systems?

When confronting contradictory data about DIS3L2 function:

• Systematic comparison of model systems:

  • Compare DIS3L2 function across multiple model systems (human cells, Drosophila, mouse)

  • Account for potential species-specific differences in DIS3L2 targets or interacting partners

  • Consider tissue-specific functions, as DIS3L2 may have different roles in different cell types

• Technical considerations:

  • Compare knockout versus knockdown approaches, as residual DIS3L2 activity may complicate interpretation

  • Evaluate the specificity of tools used (antibodies, siRNAs, CRISPR guides)

  • Consider the timing of analyses, as acute versus chronic DIS3L2 depletion may yield different results

• Contextual dependencies:

  • Investigate whether DIS3L2 function depends on cell state (proliferating vs. differentiated)

  • Test functions under various stress conditions (ER stress, nutrient limitation)

  • Consider developmental stage-specific effects

• Integration of diverse data types:

  • Combine genomic, transcriptomic, and proteomic analyses for a comprehensive view

  • Use computational approaches to identify common pathways across different experimental systems

  • Develop unified models that can explain seemingly contradictory observations