LARS Antibody

What is LARS and what is its biological significance?

LARS (leucyl-tRNA synthetase) is an essential enzyme responsible for charging tRNA molecules with leucine amino acids during protein synthesis. It has a calculated molecular weight of 134 kDa and consists of 1176 amino acids . LARS plays a critical role in translation by ensuring the correct attachment of leucine to its corresponding tRNA molecules, particularly tRNA-Leu CAG, tRNA-Leu AAG, and tRNA-Leu UAG . Beyond its canonical role in protein synthesis, recent research has identified additional functions, including its unexpected role as a tumor suppressor in breast cancer . This dual functionality makes LARS an intriguing target for cancer research.

What applications are LARS antibodies validated for?

LARS antibodies have been validated for multiple research applications as outlined in the table below:

These diverse applications enable comprehensive investigation of LARS expression, localization, and interactions in experimental systems .

Which cell lines are recommended for LARS antibody validation?

Multiple cell lines have been validated for LARS antibody use, providing researchers with options across species and tissue types:

For polyclonal antibody 21146-1-AP:

• Human cell lines: A549 (lung adenocarcinoma), HeLa (cervical cancer), Jurkat (T lymphocyte)

For monoclonal antibody 67940-1-Ig:

• Human cell lines: HeLa, HEK-293, HepG2, Jurkat, K-562

• Rodent cell lines: PC-12 (rat), NIH/3T3 (mouse), 4T1 (mouse)

When validating a new experimental system, researchers should first confirm LARS detection in these established cell lines before proceeding to their model of interest.

What are the recommended dilutions for different applications?

Optimal antibody dilutions vary by application and specific antibody:

For 21146-1-AP (polyclonal):

• Western Blot: 1:500-1:1000

• Immunoprecipitation: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Immunohistochemistry: 1:100-1:400

• Immunofluorescence/ICC: 1:50-1:500

For 67940-1-Ig (monoclonal):

• Western Blot: 1:5000-1:50000 (higher dilution indicates greater sensitivity)

• Immunofluorescence/ICC: 1:200-1:800

It is recommended to titrate antibodies in each experimental system to determine optimal conditions. Sample-dependent variation may necessitate adjustment of these ranges to obtain optimal signal-to-noise ratios.

How is LARS protein detected in Western blot experiments?

For optimal detection:

• Use 10% SDS-PAGE for effective separation

• Transfer to nitrocellulose membranes (e.g., Amersham Protran)

• Incubate with primary antibody (21146-1-AP at 1:1000 or 67940-1-Ig at 1:5000) for 2 hours

• Use appropriate detection system (e.g., Odyssey CLx Infrared Imaging System)

• Include appropriate loading control (e.g., GAPDH)

Multiple cell lines have been validated for detection, including A549, HeLa, Jurkat, HEK-293, and others, making these suitable positive controls for new experimental systems.

How is LARS expression altered in different cancer types?

Research has revealed intriguing and sometimes contradictory patterns of LARS expression across cancer types:

In breast cancer:

• LARS becomes surprisingly repressed during mammary cell transformation

• Lower LARS mRNA and protein expression in MDA-MB-231, HCC1806, and T47D cancer cell lines compared to non-transformed MCF10A cells

• Reduced LARS levels in murine breast cancer lines 4T07 and EO771 compared to non-transformed NMuMG cells

• Copy number assays in MCF10A and HCC1806 cells suggest genomic loss of LARS

This unexpected pattern suggests LARS may function as a tumor suppressor in breast cancer, contrary to the conventional expectation that translation machinery components promote cancer growth. This finding reveals the complex, context-dependent role of LARS in oncogenesis and highlights the importance of cancer-specific investigation of LARS function.

What methodological approaches can assess LARS function in cancer?

Multiple complementary methodologies can comprehensively evaluate LARS function in cancer:

Genetic Screening and Manipulation:

• CRISPR-Cas9 genome-wide screening for identifying LARS as an essential gene (used in osteosarcoma research)

• Conditional knockout models (e.g., MMTV-Cre with floxed Lars alleles)

• RNA interference (shRNA, siRNA) for targeted knockdown

• CRISPRi for transcriptional repression of LARS or specific tRNAs

Functional Assessment:

• Cell proliferation assays (e.g., CCK8) to measure growth effects

• Colony formation assays (soft agar) to assess transformation potential

• Tumor growth studies in animal models (e.g., PyMT breast cancer model)

Molecular Analysis:

• RT-qPCR for mRNA expression using validated primers:

  • LARS-forward: 5′-ATGGCGGAAAGAAAAGGAACAG-3′

  • LARS-reverse: 5′-CAGGCCAAAGGGAAACAGACAAC-3′

• Western blot for protein expression quantification

• tRNA charging assays to assess enzymatic function

• Ribosome profiling to analyze translation efficiency and codon-specific effects

Integration of these approaches enables comprehensive characterization of LARS function in cancer biology.

How does LARS depletion affect translation and protein synthesis?

LARS depletion produces specific and selective effects on translation, particularly affecting leucine-rich transcripts:

Changes in tRNA Status:

• Reduction in the ratio of charged to total tRNA

• Decreased abundance of both charged and total tRNA-Leu CAG, tRNA-Leu AAG, and tRNA-Leu UAG

Altered Translation Dynamics:

• Increased ribosome dwell time specifically over leucine codons

• Leucine codons show significantly greater dwell time increases compared to other codons

• Negative correlation between CUG codon content and translation efficiency ratio (logTER)

Transcript-Specific Effects:

• mRNAs enriched for leucine codons (especially CUG) show reduced translation efficiency

• This creates selective pressure on the proteome based on codon usage patterns

These findings reveal that LARS depletion does not uniformly affect all protein synthesis but creates a selective translational stress that preferentially impacts leucine-rich transcripts, potentially altering the cellular proteome to favor specific phenotypes such as increased proliferation in breast cancer.

What evidence supports LARS as a tumor suppressor in breast cancer?

Multiple lines of evidence support LARS's unexpected role as a tumor suppressor in breast cancer:

Genetic Evidence:

• Monoallelic deletion of LARS (reducing protein levels by ~50%) significantly increased tumor number and burden in the PyMT mouse model

• LARS-depleted tumors showed enhanced proliferation as evidenced by increased Ki67 staining

Expression Patterns:

• Reduced LARS mRNA and protein in multiple human breast cancer cell lines (MDA-MB-231, HCC1806, T47D) compared to non-transformed MCF10A cells

• Similarly reduced expression in mouse breast cancer lines (4T07, EO771) relative to normal mammary epithelial cells

• Copy number assays suggest genomic loss of LARS in breast cancer cell lines

Mechanistic Insights:

• LARS depletion reduces charging of specific tRNA-Leu isoacceptors

• CRISPRi-mediated depletion of tRNA-Leu CAG enhanced transformation of mammary epithelial cells

• This identifies tRNA-Leu CAG as a downstream tumor suppressor

These findings highlight a complex regulatory role for LARS in breast cancer where its reduction appears to promote oncogenic processes, challenging conventional understanding of translational regulation in cancer.

How can CRISPR-Cas9 screening identify essential genes like LARS?

CRISPR-Cas9 screening provides a systematic approach for identifying essential genes like LARS in cancer contexts:

Screening Methodology:

• Design of genome-wide CRISPR libraries targeting most human genes

• Transduction of cancer cells with the library

• Selection period allowing essential gene phenotypes to manifest

• Collection of surviving cells and quantification of guide RNA abundance

• Statistical comparison to identify depleted guides targeting essential genes

As demonstrated in osteosarcoma research, this approach identified LARS as an essential gene using the DepMap database, which aggregates CRISPR screening data across multiple cancer types .

Validation Workflow:

• Secondary screening with focused libraries

• Individual knockout experiments

• Functional assays (e.g., CCK8 for proliferation)

• Expression analysis in patient samples (e.g., GSE19276 database)

This systematic approach enables unbiased identification of genes critical for cancer cell survival, revealing LARS as essential despite its potential tumor suppressor role in other cancer types, highlighting context-dependent functions.

What is the optimal protocol for LARS antibody Western blotting?

For optimal Western blot results with LARS antibodies, follow this detailed protocol:

Sample Preparation:

• Lyse cells in RIPA buffer containing protease inhibitors

• Quantify protein using Micro BCA Protein Assay Kit

• Separate 20-40 μg protein on 10% SDS-PAGE gels

• Transfer to nitrocellulose membranes (e.g., Amersham Protran)

Primary Antibody Incubation:

• For 21146-1-AP: Use 1:1000 dilution for 2 hours at room temperature

• For 67940-1-Ig: Use 1:5000-1:10000 dilution for enhanced sensitivity

• Include appropriate loading control (e.g., GAPDH at 1:5000)

Detection and Quantification:

• Use infrared-based detection systems (e.g., Odyssey CLx) for precise quantification

• Alternatively, use enhanced chemiluminescence

• For quantification, normalize LARS signal to loading control

Expected Results:

• LARS protein should appear at 135-140 kDa

• Signal should be detectable in validated cell lines (A549, HeLa, Jurkat, etc.)

• Little to no background in knockout/knockdown controls

This protocol ensures reliable detection and quantification of LARS protein across experimental systems.

How can LARS expression be effectively measured at mRNA level?

For comprehensive analysis of LARS mRNA expression:

RNA Isolation and Quality Control:

• Extract total RNA using TRIzol or equivalent reagent

• Assess RNA quality (e.g., Bioanalyzer, gel electrophoresis)

• Perform DNase treatment to remove genomic DNA contamination

RT-qPCR Protocol:

• Reverse transcribe using a reliable mRNA reverse transcription kit (e.g., Takara)

• Perform qPCR with LARS-specific primers:

  • Forward: 5′-ATGGCGGAAAGAAAAGGAACAG-3′

  • Reverse: 5′-CAGGCCAAAGGGAAACAGACAAC-3′

• Use stable reference genes like GAPDH:

  • Forward: 5′-GCGGGGCTCTCCAGAACATCAT-3′

  • Reverse: 5′-CCAGCCCCAGCGTCAAAGGTG-3′

• Calculate relative expression using the 2^-ΔΔCt method

Validation and Controls:

• Include no-template and no-RT controls

• Verify primer efficiency using standard curves

• Use positive control samples (e.g., normal tissue or cell lines)

• Include LARS knockdown/knockout samples as negative controls

This methodology provides reliable quantification of LARS mRNA expression across experimental conditions and cell types.

What considerations are important for LARS knockdown experiments?

When designing LARS knockdown experiments, several critical considerations ensure robust and interpretable results:

Knockdown Approach Selection:

• siRNA: For transient knockdown with minimal off-target effects

• shRNA: For stable knockdown in long-term experiments

• CRISPR-Cas9: For complete knockout (note that complete loss may be lethal)

• CRISPRi: For transcriptional repression (useful for partial reduction)

• Conditional systems: For inducible or tissue-specific knockdown (e.g., Cre-loxP)

Knockdown Verification:

• Confirm reduction at mRNA level by RT-qPCR

• Validate protein reduction by Western blot

• Target 50-80% reduction for functional studies (complete loss may be lethal)

Experimental Design:

• Consider monoallelic knockdown (50% reduction was sufficient to observe phenotypes in breast cancer models)

• Include appropriate controls (non-targeting siRNA/shRNA, empty vector)

• Perform rescue experiments to confirm specificity

• Assess cell-type specific responses (different cancer types may respond differently)

Phenotypic Analysis:

• Proliferation assessment (e.g., CCK8 assay)

• tRNA charging effects (examine leucyl-tRNA pools)

• Translation efficiency (global and transcript-specific)

• Transformation potential (e.g., soft agar colony formation)

These considerations ensure meaningful results when manipulating LARS expression in experimental systems.

How can ribosome profiling reveal LARS depletion effects?

Ribosome profiling provides mechanistic insights into how LARS depletion affects translation:

Experimental Workflow:

• Generate LARS-depleted cells alongside controls

• Prepare ribosome-protected fragments (RPFs) by nuclease digestion

• Isolate and sequence RPFs along with total mRNA

• Analyze translation efficiency and ribosome positioning

Key Analytical Approaches:

• Translation Efficiency Analysis:

  • Calculate the ratio of ribosome occupancy to mRNA abundance

  • Compare between LARS-depleted and control cells

  • Identify transcripts most affected by LARS depletion

• Codon-Specific Analysis:

  • Calculate codon bias coefficients to measure ribosome dwell time

  • Compare dwell times over leucine codons versus other codons

  • Previous studies found significantly increased dwell time over leucine codons in LARS-depleted cells

• Transcript Feature Analysis:

  • Correlate translation effects with leucine content and codon usage

  • Previous studies showed negative correlation between CUG codon enrichment and translation efficiency in LARS-depleted cells

This approach has revealed that LARS depletion creates selective translational stress focused on leucine-rich transcripts, providing mechanistic insight into the cellular consequences of LARS reduction.

What are best practices for immunofluorescence with LARS antibodies?

For optimal immunofluorescence results with LARS antibodies:

Cell Preparation:

• Grow cells on coverslips or chamber slides (30-50% confluence)

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

• Permeabilize with 0.1-0.5% Triton X-100 in PBS

• Block with 1-5% BSA or normal serum in PBS

Antibody Application:

• For 21146-1-AP: Use 1:50-1:500 dilution

• For 67940-1-Ig: Use 1:200-1:800 dilution

• Incubate overnight at 4°C or 1-2 hours at room temperature

• Validated in multiple cell lines, particularly HeLa cells

Controls and Validation:

• Include secondary-only negative controls

• Use positive control cell lines (HeLa, Jurkat)

• Consider LARS knockdown cells as specificity controls

• Include co-staining with markers of relevant subcellular structures

Imaging and Analysis:

• Use confocal microscopy for detailed subcellular localization

• Capture z-stacks for complete visualization

• Quantify signal intensity using appropriate image analysis software

• Compare localization patterns across different experimental conditions

Following these guidelines ensures reliable and reproducible visualization of LARS localization in cellular contexts.