LAS1L Antibody

What is LAS1L and why is it important in cellular function?

LAS1L (LAS1-like ribosome biogenesis factor) is a nucleolar protein essential for cell proliferation and ribosome biogenesis. In humans, the canonical protein has 734 amino acid residues with a molecular mass of 83.1 kDa . LAS1L is primarily localized in the nucleus and cytoplasm, with critical functions in the nucleolus. It belongs to the LAS1 protein family and plays a crucial role in the biogenesis of the 60S ribosomal subunit . LAS1L's importance is highlighted by the fact that its depletion leads to inhibition of rRNA processing, impaired synthesis of mature 28S rRNA, and ultimately affects cell proliferation through disruption of ribosome formation .

What types of LAS1L antibodies are available for research?

Several types of LAS1L antibodies are available for research applications, including both monoclonal and polyclonal variants. The most common forms are unconjugated antibodies, though some suppliers offer conjugated versions for specialized applications . Both rabbit and mouse-derived antibodies are available, with options like rabbit monoclonal anti-LAS1L antibody [EPR8988(B)] from suppliers like Abcam, which has been cited in published research . The reactivity of these antibodies varies, with most targeting human LAS1L, while some also recognize mouse and rat orthologs .

What are the basic applications for LAS1L antibodies in research?

LAS1L antibodies are primarily used in several key applications:

• Western Blot (WB): The most widely used application for detecting LAS1L protein expression and measuring protein levels in various experimental conditions .

• Enzyme-Linked Immunosorbent Assay (ELISA): Used for quantitative analysis of LAS1L expression .

• Immunocytochemistry (ICC) and Immunofluorescence (IF): Used to visualize the subcellular localization of LAS1L, particularly its nucleolar localization .

• Immunoprecipitation (IP): Used to study protein-protein interactions involving LAS1L .

These techniques have been instrumental in establishing LAS1L's role in ribosome biogenesis and understanding its interaction network.

How should I design experiments to study LAS1L's role in ribosome biogenesis?

When designing experiments to study LAS1L's role in ribosome biogenesis, consider using a multi-faceted approach:

• RNA interference (RNAi): Design experiments using shRNA or siRNA to deplete LAS1L. Previous studies have used sequences like 5'-CCGGGCTGCTACTTTGTCCTGGATTCTCGAGAATCCAGGACAAAGTAGCAGCTTTTTTG-3' cloned into pLKO.1-puro lentiviral vectors for effective knockdown .

• rRNA processing analysis: Monitor pre-rRNA processing after LAS1L depletion, with particular focus on the 32S pre-rRNA intermediate, which accumulates in LAS1L-depleted cells, indicating processing defects in the internal transcribed spacer-2 (ITS-2) .

• Cell cycle analysis: Include BrdU labeling with propidium iodide staining for FACS analysis to detect the G1 cell cycle arrest that occurs upon LAS1L depletion .

• p53 pathway analysis: Monitor p53 and p21 levels through Western blotting to confirm the nucleolar stress response induced by LAS1L depletion .

• Nucleolar integrity assessment: Use immunofluorescence with antibodies against nucleolar markers like UBF, fibrillarin, and nucleophosmin alongside LAS1L antibodies to assess nucleolar disruption .

What controls should I include when using LAS1L antibodies in immunofluorescence studies?

When conducting immunofluorescence studies with LAS1L antibodies, include the following controls:

• Negative controls:

  • Primary antibody omission: Perform the immunofluorescence protocol without the primary LAS1L antibody to assess background from secondary antibodies.

  • RNAi-depleted samples: Include cells where LAS1L has been depleted through RNAi to confirm antibody specificity .

  • Isotype controls: Use an irrelevant antibody of the same isotype as your LAS1L antibody.

• Positive controls:

  • Co-staining with established nucleolar markers: Use antibodies against UBF, fibrillarin, or nucleophosmin, which should partially colocalize with LAS1L in the nucleolus .

  • Overexpression controls: Include cells transfected with LAS1L expression constructs.

• Technical controls:

  • Fixed cell samples should be permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature to ensure antibody access to nuclear antigens .

  • Block samples with 1% BSA for 30 minutes to reduce non-specific binding .

  • Use appropriate fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488 goat anti-rabbit or Alexa Fluor 647 goat anti-mouse) .

What is the optimal protocol for detecting LAS1L in Western blot experiments?

For optimal detection of LAS1L in Western blot experiments, follow these methodological guidelines:

• Sample preparation:

  • Extract total protein from your samples using a buffer containing protease inhibitors.

  • For nuclear proteins like LAS1L, consider using nuclear extraction protocols for enrichment.

• Gel selection:

  • Use 8-10% SDS-PAGE gels to properly resolve the 83.1 kDa LAS1L protein .

• Transfer conditions:

  • Transfer proteins to PVDF or nitrocellulose membranes using either wet or semi-dry transfer systems.

• Blocking and antibody incubation:

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

  • Incubate with primary LAS1L antibody at manufacturer-recommended dilutions (typically 1:1000) overnight at 4°C.

  • Wash thoroughly with TBST before incubating with appropriate HRP-conjugated secondary antibody.

• Detection:

  • Use enhanced chemiluminescence (ECL) for detection of the specific LAS1L band at approximately 83.1 kDa .

• Controls:

  • Include positive controls (cell lines known to express LAS1L).

  • Include negative controls (LAS1L-depleted cells through RNAi) .

  • Consider using loading controls such as β-actin or GAPDH for normalization.

How can I investigate LAS1L protein interactions and complexes?

To investigate LAS1L protein interactions and complexes, consider these advanced approaches:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate endogenous LAS1L using validated antibodies and identify associated proteins by Western blotting or mass spectrometry .

  • Previous studies have identified six proteins associated with LAS1L through this approach: PELP1, RanBP5, TEX10, NOL9, SENP3, and WDR18 .

• Proximity-based labeling:

  • Consider BioID or APEX2-based approaches to identify proteins in close proximity to LAS1L in living cells.

• Sucrose gradient fractionation:

  • Use this technique to separate ribosomal subunits and pre-ribosomal particles, then detect LAS1L in different fractions to understand its association with specific ribosomal assembly intermediates.

• Chromatin Immunoprecipitation (ChIP):

  • If investigating LAS1L's potential role in transcriptional regulation, perform ChIP experiments to identify genomic binding sites.

• CRISPR-Cas9 gene editing:

  • Generate cell lines with tagged endogenous LAS1L (e.g., FLAG, HA, or GFP tags) for purification of native complexes.

• Validation of interactions:

  • Confirm identified interactions through reciprocal Co-IPs and depletion studies to assess the functional significance of each interaction .

What methods are best for studying the role of LAS1L in rRNA processing?

To effectively study LAS1L's role in rRNA processing, employ the following specialized methods:

• Northern blot analysis:

  • Design probes targeting specific regions of pre-rRNA and mature rRNAs to detect processing intermediates that accumulate upon LAS1L depletion.

  • Focus particularly on the 32S pre-rRNA intermediate, which accumulates when LAS1L function is compromised .

• Pulse-chase labeling:

  • Use metabolic labeling with 32P or similar radioisotopes to track the kinetics of rRNA processing in control versus LAS1L-depleted cells.

• rRNA quantification by qRT-PCR:

  • Design primers specific to various pre-rRNA regions and mature rRNAs to quantify processing efficiency.

  • Amplify exons 5-10 in the LAS1L transcript for expression analysis .

• Fluorescence in situ hybridization (FISH):

  • Use probes targeting pre-rRNA species to visualize their localization and abundance in control versus LAS1L-depleted cells.

• Ribosome profiling:

  • Analyze translational effects of LAS1L depletion through ribosome profiling to understand the functional consequences of disrupted ribosome biogenesis.

• Ribosomal subunit analysis:

  • Use sucrose gradient centrifugation to quantify 40S and 60S ribosomal subunits and 80S monosomes in control versus LAS1L-depleted cells.

How can I investigate the relationship between LAS1L dysfunction and human disease?

To investigate links between LAS1L dysfunction and human disease, consider these research approaches:

• Mutation analysis:

  • Screen for LAS1L mutations in patients with intellectual developmental disorders, as LAS1L has been associated with these conditions .

  • Specifically investigate potential connections to Wilson-Turner syndrome (WTS) and infantile hypotonia with respiratory failure .

• Patient-derived cells:

  • Establish cell lines from patients with suspected LAS1L-related disorders to study protein expression, localization, and function.

• CRISPR-Cas9 disease modeling:

  • Generate cell lines or model organisms with specific patient-derived LAS1L mutations to study their functional consequences.

• Structure-function studies:

  • Map disease-associated mutations onto the functional domains of LAS1L to understand how they disrupt protein function.

• Transcriptome and proteome analysis:

  • Compare gene expression and protein profiles between normal and LAS1L-mutant or depleted cells to identify dysregulated pathways.

• Cell-based assays:

  • Develop rescue experiments where wildtype LAS1L is re-introduced into patient cells to determine if cellular phenotypes can be reversed.

Why might I be observing non-specific bands in LAS1L Western blots?

Non-specific bands in LAS1L Western blots can arise from several sources:

• Isoform detection:

  • LAS1L has up to 4 different reported isoforms , which might appear as additional bands. Compare observed band sizes with predicted isoform molecular weights.

• Post-translational modifications:

  • LAS1L may undergo phosphorylation, ubiquitination, or other modifications that alter its migration pattern.

• Antibody specificity issues:

  • Some antibodies may cross-react with related proteins. Validate specificity by:

    • Using LAS1L-depleted samples as negative controls

    • Testing multiple antibodies targeting different epitopes

    • Performing peptide competition assays

• Sample preparation problems:

  • Protein degradation during extraction can produce fragments that appear as additional bands

  • Incomplete denaturation can lead to aggregates or multimers

• Technical solutions:

  • Increase washing stringency (higher salt or detergent concentration)

  • Optimize antibody dilution

  • Consider using monoclonal antibodies for higher specificity

  • Pre-absorb antibodies with common cross-reactive proteins

What are the potential pitfalls in using RNAi to study LAS1L function?

When using RNAi to study LAS1L function, be aware of these potential pitfalls:

• Incomplete knockdown:

  • LAS1L is essential for cell proliferation, and cells with complete knockdown may be selected against, resulting in mixed populations with variable depletion levels.

  • Solution: Use inducible shRNA systems or optimize transfection/transduction efficiency.

• Off-target effects:

  • siRNAs or shRNAs may affect expression of genes other than LAS1L.

  • Solution: Use multiple independent RNAi sequences targeting different regions of LAS1L and perform rescue experiments with RNAi-resistant LAS1L constructs .

• Secondary effects versus direct effects:

  • LAS1L depletion causes cell cycle arrest and p53 activation , which can confound interpretation of experimental results.

  • Solution: Use p53-null cell lines or p53 co-depletion to distinguish direct roles of LAS1L from secondary effects of p53 activation.

• Timing considerations:

  • Acute versus chronic depletion may yield different phenotypes.

  • Solution: Perform time-course experiments to distinguish primary from secondary effects.

• Technical validation:

  • Always confirm knockdown efficiency at both mRNA level (using qRT-PCR) and protein level (using Western blot) .

How can I distinguish between LAS1L's direct roles and indirect effects in ribosome biogenesis?

Distinguishing direct from indirect effects of LAS1L in ribosome biogenesis requires careful experimental design:

• Temporal analysis:

  • Perform time-course experiments after LAS1L depletion to identify which defects appear first (likely direct effects) versus later (potentially secondary).

• Structure-function studies:

  • Generate mutant LAS1L constructs affecting different functional domains to dissect which activities are essential for which aspects of ribosome biogenesis.

• Protein-RNA interaction studies:

  • Use RNA immunoprecipitation (RIP) or crosslinking immunoprecipitation (CLIP) to identify direct RNA targets of LAS1L.

• Proximity labeling approaches:

  • Employ BioID or APEX2 fusions with LAS1L to identify proteins in close proximity during ribosome synthesis.

• In vitro reconstitution:

  • Attempt to reconstitute specific steps of ribosome assembly with purified components including LAS1L to test direct biochemical roles.

• Conditional depletion systems:

  • Use rapid protein degradation systems (e.g., auxin-inducible degron) to achieve acute LAS1L depletion and observe immediate consequences.

What is known about LAS1L mutations in human diseases?

LAS1L mutations have been implicated in several human diseases:

• Intellectual developmental disorders:

  • The LAS1L gene has been associated with intellectual developmental disorders, suggesting a critical role in neurodevelopment .

• Wilson-Turner syndrome (WTS):

  • Mutations in LAS1L can be associated with Wilson-Turner syndrome, a rare X-linked condition characterized by intellectual disability, obesity, gynecomastia, emotional lability, and tapering fingers .

• Severe infantile conditions:

  • More rarely, LAS1L mutations can cause severe infantile hypotonia with respiratory failure, representing a more acute manifestation of LAS1L dysfunction .

• Potential cancer connections:

  • Given LAS1L's role in ribosome biogenesis and the link between dysregulated ribosome synthesis and cancer, further investigation into potential cancer associations is warranted, though direct evidence is currently limited.

• Research approaches:

  • Genetic screening of affected populations

  • Functional characterization of patient-derived mutations

  • Animal models of LAS1L-associated diseases

How can advanced molecular techniques improve our understanding of LAS1L function?

Emerging molecular techniques that could advance LAS1L research include:

• Cryo-electron microscopy:

  • Determine the structure of LAS1L alone and in complex with associated proteins and RNA to understand molecular mechanisms of action.

• Integrative structural biology:

  • Combine X-ray crystallography, NMR, mass spectrometry, and computational modeling to build comprehensive structural models of LAS1L complexes.

• Single-molecule approaches:

  • Use techniques like single-molecule FRET to study conformational changes and dynamics of LAS1L during ribosome assembly.

• Spatial transcriptomics:

  • Apply techniques to visualize and quantify rRNA processing intermediates in situ to understand the spatial organization of ribosome assembly.

• Proteomics:

  • Apply quantitative proteomics to identify changes in the composition of pre-ribosomal particles upon LAS1L depletion or mutation.

• CRISPR screening:

  • Perform genome-wide CRISPR screens in LAS1L-depleted versus control cells to identify genetic interactions and compensatory pathways.

What are potential therapeutic approaches for diseases associated with LAS1L dysfunction?

While therapeutic approaches for LAS1L-associated disorders are still in early stages, several potential strategies could be considered:

• Gene therapy approaches:

  • For loss-of-function mutations, delivery of functional LAS1L cDNA could potentially restore normal protein function.

• RNA-based therapeutics:

  • Antisense oligonucleotides or small interfering RNAs could be designed to modulate expression of specific LAS1L isoforms or target downstream effectors.

• Small molecule screening:

  • Identify compounds that could stabilize mutant LAS1L protein or enhance residual function.

• Targeting downstream pathways:

  • Identify and modulate pathways dysregulated as a consequence of LAS1L dysfunction, particularly focusing on p53-dependent responses .

• Ribosome-targeted therapies:

  • Explore the potential of compounds that modulate ribosome biogenesis or function to compensate for LAS1L dysfunction.

• Personalized medicine approaches:

  • Develop patient-derived cellular models to test therapeutic strategies tailored to specific LAS1L mutations.