LARS Antibody, FITC conjugated

Basic Research Questions

• What is LARS antibody and what are its primary research applications?

LARS antibody targets leucyl-tRNA synthetase, an essential enzyme responsible for attaching leucine to its cognate tRNA during protein synthesis. The full-length human LARS protein consists of 1176 amino acids with a calculated molecular weight of 134 kDa, though it typically appears at 135-140 kDa on Western blots .

LARS antibodies are employed in various research applications including:

When conjugated with FITC, these antibodies are particularly useful for flow cytometry and fluorescence microscopy applications .

• How does FITC conjugation of LARS antibody enhance its research utility?

FITC (Fluorescein isothiocyanate) conjugation provides LARS antibodies with direct fluorescent detection capabilities, eliminating the need for secondary antibodies in many applications. The conjugation process involves covalently linking FITC molecules to primary amines on the antibody structure.

Modern conjugation kits allow this process to be completed in under 20 minutes with minimal hands-on time (approximately 30 seconds) and offer 100% antibody recovery . This direct labeling approach provides several advantages:

• Reduction of background signal by eliminating cross-reactivity from secondary antibodies

• Simplified multiplexing with other antibodies in co-staining experiments

• Direct visualization in applications like flow cytometry, fluorescence microscopy, and live cell imaging

• Enhanced detection sensitivity in samples with low LARS expression levels

• What sample types are compatible with LARS antibody-FITC conjugates?

LARS antibody has demonstrated reactivity with various sample types, with primary compatibility with human tissues and cell lines. Based on validation studies, the following sample compatibilities have been established:

For flow cytometry applications using FITC-conjugated antibodies, both human and porcine blood samples have been successfully analyzed after proper sample preparation, including erythrocyte lysis using appropriate buffers . When working with new sample types, preliminary validation experiments are recommended to determine optimal conditions.

• What are the recommended protocols for using FITC-conjugated LARS antibody in flow cytometry?

When using FITC-conjugated LARS antibody for flow cytometry, researchers should follow these methodological steps:

• Sample Preparation: Collect cells and wash in PBS containing 1% BSA at 4°C.

• Cell Fixation: Fix cells in 4% paraformaldehyde for 10-15 minutes if required. For live cell analysis, skip this step.

• Antibody Incubation: Add FITC-conjugated LARS antibody at a concentration of 5-10 μg/ml and incubate for 30 minutes at 4°C in the dark.

• Washing: Wash cells 3 times with PBS containing 1% BSA.

• Erythrocyte Lysis: For blood samples, lyse erythrocytes using commercially available lysing solutions (like BD FACS Lysing) for human samples or a solution of 0.16 M ammonium chloride, 10 mM sodium bicarbonate, 0.12 mM EDTA, and 0.04% paraformaldehyde for porcine samples .

• Analysis: Analyze using appropriate gating strategies based on forward and side scatter properties.

FITC is excited at 488 nm and emits at 520 nm, requiring appropriate laser and filter settings on the flow cytometer.

• What are the optimal storage conditions for maintaining FITC-conjugated LARS antibody activity?

To preserve the activity and fluorescence intensity of FITC-conjugated LARS antibody, proper storage conditions are essential:

• Store at -20°C in a non-frost-free freezer to prevent freeze-thaw cycles

• Protect from light exposure by using amber tubes or wrapping in aluminum foil

• Store in buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquot into single-use volumes to minimize freeze-thaw cycles

• For short-term storage (< 1 month), 4°C is acceptable if protected from light

FITC conjugates are generally stable for at least one year when stored properly . Avoid repeated freeze-thaw cycles as they can lead to protein denaturation and fluorophore degradation, resulting in decreased signal intensity and increased background.

Intermediate Research Questions

• How can I perform FITC conjugation of LARS antibody in my laboratory?

To conjugate LARS antibody with FITC in your laboratory, follow this detailed methodological approach:

• Select an appropriate conjugation kit: Lightning-Link® FITC conjugation kits offer rapid conjugation with minimal hands-on time (< 30 seconds) and complete in under 20 minutes .

• Prepare the antibody:

  • Ensure LARS antibody is in a buffer free of primary amines (e.g., avoid Tris)

  • Recommended antibody concentration: 1-4 mg/ml

  • If necessary, concentrate using ultrafiltration devices with appropriate MWCO

• Conjugation procedure:

  • Add the antibody to the Lightning-Link® FITC reagent vial

  • Add the LL-Modifier reagent (1 μl per 10 μl of antibody)

  • Mix gently and incubate at room temperature for 15 minutes

  • Add LL-Quencher reagent (1 μl per 10 μl of antibody)

• Validation: Confirm successful conjugation using spectrophotometric analysis (A280 for protein concentration and A495 for FITC)

• Calculate F/P ratio: Determine the fluorophore-to-protein ratio using the formula:
  $\text{F/P ratio} = \frac{A_{495} \times \text{dilution factor} \times \text{MW of antibody}}{195,000 \times \text{antibody concentration (mg/ml)}}$

  Optimal F/P ratios typically range from 4-7 for IgG antibodies

This approach has been successfully used to conjugate FITC to various antibodies for flow cytometry applications, including Rat anti-PDGFRalpha and anti-α-Synuclein antibodies .

• What controls should be included when using FITC-conjugated LARS antibody in immunofluorescence experiments?

A comprehensive control strategy is essential when using FITC-conjugated LARS antibody in immunofluorescence experiments:

Essential Controls:

• Isotype Control: Use a FITC-conjugated antibody of the same isotype (e.g., Rabbit IgG-FITC for LARS polyclonal antibody) to assess non-specific binding.

• Negative Cell/Tissue Control: Include samples known to be negative for LARS expression.

• Positive Cell/Tissue Control: Include validated positive samples such as HeLa cells for IF/ICC .

• Unstained Sample: To establish autofluorescence baseline.

• Single-Color Controls: When multiplexing, include single-color samples to set compensation parameters.

Advanced Controls:

• Blocking Peptide Control: Pre-incubate FITC-LARS antibody with LARS immunogen peptide to confirm specificity.

• siRNA Knockdown: Analyze samples with LARS knockdown to validate antibody specificity.

• Secondary-Only Control: If using indirect methods, include samples with secondary antibody only.

• Fixation Control: Compare live versus fixed samples to assess effects of fixation on epitope recognition.

These controls should be processed identically to experimental samples and imaged using the same acquisition parameters to ensure valid comparisons.

• How does LARS antibody-FITC perform in combination with other fluorophore-conjugated antibodies?

LARS antibody-FITC can be effectively used in multiplex immunofluorescence assays with other fluorophore-conjugated antibodies, provided proper experimental design:

Spectral Considerations:

• FITC excitation maximum: 495 nm

• FITC emission maximum: 520 nm

• Compatible fluorophores with minimal spectral overlap include:

  • Cy5 (649/670 nm)

  • Alexa Fluor 647 (650/668 nm)

  • PE-Cy7 (496/785 nm)

  • APC (650/660 nm)

Optimization Strategies:

• Sequential Staining: For challenging multiplexing, apply antibodies sequentially rather than simultaneously

• Titration: Determine optimal concentrations for each antibody individually before multiplexing

• Blocking: Use species-specific blocking reagents to prevent cross-reactivity

• Compensation: Apply appropriate spectral compensation when analyzing by flow cytometry

Example Multiplex Panel:
Flow cytometry studies have successfully used FITC-conjugated antibodies in combination with other markers. For instance, one study utilized a multi-color panel to identify oligodendroglial cells with FITC-conjugated anti-PDGFRα alongside markers like A2B5, NG2, O4, MOG, and GALC .

• What are the common challenges in preparing samples for LARS-FITC analysis by immunohistochemistry?

When preparing samples for LARS-FITC analysis by immunohistochemistry, researchers should address several technical challenges:

Fixation Challenges:

• Overfixation can mask epitopes, while underfixation may compromise tissue morphology

• Recommendation: Use 4% paraformaldehyde fixation for 24-48 hours at 4°C for optimal results

Antigen Retrieval Requirements:
For LARS detection in human colon tissue, heat-induced epitope retrieval is required:

• Preferred method: TE buffer at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

Autofluorescence Mitigation:

• Treatment with 0.1% sodium borohydride for 5 minutes reduces fixative-induced autofluorescence

• For tissues with high autofluorescence (e.g., brain), use Sudan Black B (0.1% in 70% ethanol) for 10 minutes

Section Thickness Optimization:

• For FFPE tissues: 4-6 μm sections are optimal

• For frozen tissues: 8-10 μm sections provide better signal-to-noise ratio

Blocking Protocol:

• Block with 5-10% normal serum from the same species as the secondary antibody

• Add 0.1-0.3% Triton X-100 for membrane permeabilization

• Include 0.1% BSA to reduce non-specific binding

For human colon tissue, the validated dilution for LARS antibody in IHC is 1:100-1:400 , which may require adjustment when using the FITC-conjugated version.

• How can I validate the specificity of a newly conjugated LARS antibody-FITC?

To thoroughly validate the specificity of a newly conjugated LARS antibody-FITC, implement this systematic approach:

Analytical Validation Methods:

• Western Blot Comparison:

  • Compare staining pattern of unconjugated versus FITC-conjugated LARS antibody

  • Verify correct molecular weight (135-140 kDa)

  • Test in validated positive cell lines: A549, HeLa, Jurkat cells

• Immunoprecipitation:

  • Perform IP with unconjugated and FITC-conjugated antibody

  • Confirm similar pull-down efficiency in Jurkat cells

• Knockdown/Knockout Validation:

  • Test antibody in LARS-knockdown or knockout samples

  • Published studies have used KD/KO approaches for LARS antibody validation

• Competitive Binding Assay:

  • Pre-incubate with excess LARS immunogen peptide

  • Confirm signal reduction in positive samples

• Flow Cytometric Analysis:

  • Compare staining patterns with established LARS antibodies

  • Evaluate in parallel with isotype control

  • Analyze cell populations expected to express LARS

• Cross-Reactivity Assessment:

  • Test in samples from multiple species if cross-reactivity is expected

  • LARS antibodies have shown reactivity with human and mouse samples

Document all validation results comprehensively, including images, experimental conditions, and statistical analyses to establish confidence in antibody specificity.

Advanced Research Questions

• What strategies can optimize signal-to-noise ratio when using FITC-conjugated LARS antibody in flow cytometry?

Optimizing signal-to-noise ratio for FITC-conjugated LARS antibody in flow cytometry requires addressing several technical parameters:

Sample Preparation Optimization:

• Maintain cells at 4°C during processing to prevent endocytosis and capping

• Use viability dyes to exclude dead cells, which can non-specifically bind antibodies

• Apply RNase/DNase treatment for nucleated cells with high RNA/DNA content

Signal Enhancement Approaches:

• F/P Ratio Optimization: Determine optimal fluorophore-to-protein ratio (typically 4-7 for FITC)

• Fluorophore Selection: Consider brighter alternatives to FITC (e.g., Alexa Fluor 488) for low abundance targets

• Amplification Systems: Implement tyramide signal amplification for weak signals

Background Reduction Techniques:

• Blocking Strategy: Use 5% BSA with 5-10% serum matching secondary antibody species

• Fc Receptor Blocking: Pre-treat cells with Fc block (anti-CD16/CD32) for 15 minutes

• Autofluorescence Reduction: Use specific buffers or dyes that quench cellular autofluorescence

Instrument Settings Optimization:

• PMT Voltage: Set optimal voltage for FITC channel using unstained and single-stained controls

• Threshold Adjustment: Apply FSC/SSC thresholds to exclude debris and aggregates

• Compensation: Properly compensate for spectral overlap with other fluorophores

Analysis Algorithm Enhancement:

• Spreading Error Reduction: Apply fluorescence-minus-one (FMO) controls

• Alternative Analysis: Consider biexponential or logicle display for better resolution

• Population Identification: Use hierarchical gating strategies for accurate population definition

Researchers have successfully applied these strategies when using FITC-conjugated antibodies for detecting various cell populations in complex samples like spinal cord and blood .

• How can LARS antibody-FITC be used to investigate protein-protein interactions in leucyl-tRNA synthetase complexes?

Investigating protein-protein interactions involving LARS using FITC-conjugated antibodies requires sophisticated methodological approaches:

Proximity-Based Detection Methods:

• FRET Analysis:

  • Conjugate potential interaction partners with complementary fluorophores (FITC on LARS, acceptor fluorophore on partner protein)

  • Measure energy transfer to detect interactions within 10 nm

  • Quantify FRET efficiency using acceptor photobleaching or sensitized emission

• Proximity Ligation Assay (PLA):

  • Use FITC-LARS antibody with complementary antibody against interaction partner

  • Apply oligonucleotide-conjugated secondary antibodies

  • Proximity generates circular DNA template for rolling circle amplification

  • Visualize with fluorescent probes as distinct puncta

Co-Localization Analysis Techniques:

• Confocal Microscopy:

  • Co-stain with LARS-FITC and partner protein antibodies with spectrally distinct fluorophores

  • Apply high-resolution imaging with appropriate controls

  • Quantify co-localization using Pearson's or Mander's coefficients

• Super-Resolution Microscopy:

  • Implement STORM, PALM, or STED microscopy to overcome diffraction limit

  • Achieve 20-50 nm resolution to precisely map interaction domains

  • Perform cluster analysis to identify interaction hotspots

Biochemical Validation Methods:

• Co-Immunoprecipitation:

  • Use LARS antibody (non-FITC) for pull-down

  • Detect interaction partners by Western blot

  • LARS antibody has been validated for IP and CoIP applications

• Live-Cell Protein Complementation:

  • Split fluorescent protein approach with LARS and candidate interactors

  • Visualize reconstituted fluorescence at interaction sites

  • Quantify signal intensity as measure of interaction strength

These methodologies have been successfully implemented in studies investigating protein interactions in complex cellular systems, providing insights into the molecular mechanisms of leucyl-tRNA synthetase function.

• What are the technical considerations for using LARS antibody-FITC in quantitative single-cell analysis?

Quantitative single-cell analysis using LARS antibody-FITC requires addressing several advanced technical considerations:

Standardization and Calibration:

• Fluorescence Calibration:

  • Use calibration beads with defined MESF (Molecules of Equivalent Soluble Fluorochrome) values

  • Convert fluorescence intensity to absolute molecule numbers

  • Establish standard curves relating fluorescence to antigen density

• Batch Normalization:

  • Include biological standards in each experiment

  • Apply computational normalization to correct for batch effects

  • Utilize spike-in controls for technical variation assessment

Quantitative Imaging Parameters:

• Dynamic Range Optimization:

  • Determine linear detection range for FITC signal

  • Avoid saturation through appropriate exposure settings

  • Use quality metrics like Signal-to-Noise and coefficient of variation

• Segmentation Algorithms:

  • Apply advanced cell segmentation (watershed, machine learning-based)

  • Distinguish membrane, cytoplasmic, and nuclear LARS localization

  • Extract multi-parametric features (intensity, texture, morphology)

Single-Cell Flow Cytometry Applications:

• Rare Cell Detection:

  • Process ≥1 million events for detecting rare populations

  • Implement Boolean gating strategies

  • Use backgating to confirm population identity

• Intracellular Assessment:

  • Optimize fixation and permeabilization for LARS detection

  • Balance epitope preservation and antibody accessibility

  • Use saponin (0.1%) for cytoplasmic proteins like LARS

Data Analysis Frameworks:

• High-Dimensional Analysis:

  • Apply tSNE, UMAP, or PhenoGraph for population identification

  • Perform FlowSOM or Citrus for automated population discovery

  • Implement trajectory analysis for developmental processes

• Statistical Approaches:

  • Use mixed-effects models for nested experimental designs

  • Apply appropriate tests for non-normally distributed single-cell data

  • Implement robust statistics to handle outliers

These approaches allow researchers to move beyond qualitative assessments to precisely quantify LARS expression and localization at the single-cell level, revealing biological heterogeneity often masked in bulk analyses.

• How can LARS antibody-FITC be employed in studying the relationship between leucyl-tRNA synthetase and cellular stress responses?

Investigating LARS involvement in cellular stress responses using FITC-conjugated antibodies requires sophisticated experimental approaches:

Stress Induction Models:

• Oxidative Stress:

  • Hydrogen peroxide (100-500 μM)

  • Paraquat (10-100 μM)

  • Monitor LARS localization and expression changes

• Nutrient Deprivation:

  • Leucine starvation (complete or partial)

  • Amino acid restriction

  • Track LARS redistribution between cytoplasm and nucleus

• ER Stress:

  • Tunicamycin (1-5 μg/ml)

  • Thapsigargin (100-500 nM)

  • Assess LARS interaction with stress granule markers

Methodological Approaches:

• Live-Cell Imaging with FITC-LARS:

  • Culture cells in appropriate chambers

  • Apply stress stimulus during imaging

  • Capture time-lapse images at 5-10 minute intervals

  • Quantify redistribution kinetics and co-localization with stress markers

• Flow Cytometric Stress Analysis:

  • Harvest cells at multiple timepoints post-stress

  • Measure LARS-FITC intensity in conjunction with:

    • Viability markers (Annexin V/PI)

    • Stress markers (phospho-eIF2α, ATF4)

    • ROS indicators (CM-H2DCFDA)

• Multiplex Immunofluorescence:

  • Co-stain with LARS-FITC and stress response proteins

  • Include markers for stress granules (G3BP1, TIA-1)

  • Analyze in fixed cells at defined stress timepoints

Functional Assessment Techniques:

• LARS Activity Correlation:

  • Measure aminoacylation activity in parallel with localization

  • Correlate changes in LARS-FITC distribution with enzymatic function

  • Assess impact of stress on tRNA charging efficiency

• Signaling Pathway Analysis:

  • Inhibit stress response pathways (MAPK, mTOR, PERK)

  • Track LARS-FITC localization following inhibitor treatment

  • Determine pathway dependency of LARS stress responses

These approaches have been successfully utilized to study stress-induced changes in protein localization and function, providing insights into the non-canonical roles of aminoacyl-tRNA synthetases in stress adaptation.

• What are the latest advances in using LARS antibody-FITC for detecting subcellular redistribution in disease models?

Recent advances in LARS-FITC applications for disease model subcellular redistribution studies have emerged across multiple research domains:

Neurodegenerative Disease Models:

• Parkinson's Disease:

  • α-Synuclein co-localization studies using FITC-conjugated antibodies have revealed protein interaction networks in B cells

  • Similar approaches can be applied with LARS-FITC to investigate aminoacyl-tRNA synthetase dysfunction

• Demyelinating Disorders:

  • FITC-conjugated antibodies have been used to characterize oligodendroglial populations in multiple sclerosis models

  • Application to LARS redistribution could reveal roles in protein synthesis regulation during demyelination

Methodology Enhancements:

• Multiplex Imaging Systems:

  • Integration of spectral unmixing algorithms to separate FITC signal from tissue autofluorescence

  • Implementation of cyclic immunofluorescence (CycIF) allowing 20+ markers on single samples

  • Application of artificial intelligence for automated redistribution pattern recognition

• Quantitative Subnuclear Analysis:

  • Super-resolution quantification of LARS nuclear speckle association

  • Correlation with transcriptional activity using nascent RNA labeling

  • Single-molecule tracking of LARS-FITC to identify interaction kinetics

Translational Research Applications:

• Patient-Derived Models:

  • Application of LARS-FITC in patient-derived organoids

  • Correlation of subcellular localization with disease severity

  • Identification of cell type-specific LARS distribution patterns

• Therapeutic Response Monitoring:

  • Use of LARS-FITC to track normalization of subcellular distribution following treatment

  • Integration with high-content screening for drug discovery

  • Development of redistribution metrics as pharmacodynamic biomarkers