LAMTOR1 Antibody, HRP conjugated

What is LAMTOR1 and what cellular functions does it perform?

LAMTOR1 (also known as p18, p27RF-Rho, C11orf59, PDRO) is an 18 kDa protein that acts as a key component of the pentameric Ragulator complex. It is directly responsible for anchoring the Ragulator complex to the lysosomal membrane where it coordinates metabolic signaling pathways with nutrient availability . LAMTOR1 plays multiple crucial roles in cellular function:

• Amino acid sensing and activation of mTORC1 signaling

• Regulation of lysosomal biogenesis and trafficking

• Modulation of endosomal receptor recycling

• Involvement in MAPK signaling pathway through recruitment of components to late endosomes

• Regulation of cholesterol homeostasis by controlling LDL uptake and cholesterol release from late endosomes/lysosomes

• Potential role in RHOA activation

The downregulation of LAMTOR1 affects lysosomal activation, leading to aberrant lysosomal catabolism that produces excessive reactive oxygen species, triggering p53-dependent cell cycle arrest and apoptosis .

What are the optimal applications for LAMTOR1 Antibody, HRP conjugated?

LAMTOR1 Antibody, HRP conjugated has been validated for ELISA applications . When working with this antibody, researchers should consider:

• Protein detection limit: The antibody can detect endogenous levels of LAMTOR1

• Sample types: Effective for human samples (species reactivity: Human)

• Recommended dilutions: Follow manufacturer guidelines for optimal concentration

• Detection system: Being HRP-conjugated, this antibody eliminates the need for secondary antibody incubation, providing direct colorimetric or chemiluminescent detection

For other applications such as Western Blotting, Immunoprecipitation, or Immunofluorescence, consider alternative LAMTOR1 antibodies specifically validated for those techniques .

What is the proper storage and handling protocol for LAMTOR1 Antibody, HRP conjugated?

To maintain antibody integrity and performance:

• Storage temperature: Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles which can degrade antibody quality

• Working solution preparation: Dilute only the amount needed for immediate use

• Storage buffer composition: The antibody is provided in a buffer containing 50% Glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as a preservative

• Long-term stability: Follow manufacturer's expiration date guidelines

When handling the antibody, use sterile techniques to prevent contamination and wear appropriate personal protective equipment due to the presence of Proclin 300.

How should ELISA protocols be optimized when using LAMTOR1 Antibody, HRP conjugated?

For optimal ELISA performance:

• Coating concentration: Start with standard protocols (typically 1-10 μg/ml of capture antibody)

• Blocking buffer optimization: Test different blocking agents (BSA, casein, commercial blockers) to minimize background

• Sample preparation: Ensure proper cell lysis and protein extraction methods that preserve LAMTOR1's native structure

• Antibody dilution: Perform titration experiments to determine optimal working concentration

• Incubation conditions: Optimize time (typically 1-2 hours) and temperature (room temperature or 4°C)

• Detection system: Select appropriate substrate (TMB, ABTS, or chemiluminescent substrates) based on sensitivity requirements

• Include proper controls:

  • Positive control: Recombinant LAMTOR1 protein

  • Negative control: Samples from LAMTOR1 knockout or knockdown models

  • Background control: No primary antibody

A sandwich ELISA approach might require a complementary capture antibody recognizing a different epitope of LAMTOR1.

What are the subcellular localization patterns of LAMTOR1 and how can they be visualized?

LAMTOR1 primarily localizes to:

• Lysosomal membranes (primary location)

• Late endosomes

• Plasma membrane

• Golgi apparatus

For visualization studies:

• Immunofluorescence techniques using LAMTOR1-specific antibodies

• Co-localization studies with:

  • LAMP1-YFP for lysosomes

  • Organelle-specific markers (LysoTracker for acidic compartments)

• Live-cell imaging approaches to track dynamic changes in LAMTOR1 localization

• Subcellular fractionation followed by immunoblotting to quantify LAMTOR1 distribution

When visualizing LAMTOR1, consider that its distribution may change under different cellular conditions, such as nutrient availability or stress.

How does LAMTOR1 regulate TRPML1-dependent calcium signaling and lysosomal trafficking?

LAMTOR1 directly interacts with and inhibits the transient receptor potential mucolipin 1 (TRPML1) channel, which mediates lysosomal Ca2+ release . This represents a novel function independent of mTORC1 regulation.

Methodological approaches to study this interaction:

• Structure-function analysis:

  • The N-terminal domain of LAMTOR1 (particularly residues 20-60) mediates interaction with TRPML1

  • Deletion of this domain significantly reduces LAMTOR1-TRPML1 interactions

• Functional studies in neurons:

  • LAMTOR1 knockdown increases TRPML1-mediated Ca2+ release

  • This facilitates dendritic lysosomal trafficking powered by dynein

  • Effects can be blocked by TRPML1 inhibitor ML-SI1 or TRPML1 knockdown

  • Direct activation of TRPML1 with ML-SA1 mimics LAMTOR1 knockdown effects

• Experimental paradigm:

  • Track lysosomal trafficking using LysoTracker or LAMP1-YFP labeling

  • Monitor calcium dynamics using calcium-sensitive dyes or genetically encoded calcium indicators

  • Use pharmacological tools (ML-SI1, ML-SA1) to modulate TRPML1 activity

  • Engineer LAMTOR1 constructs with domain deletions to disrupt specific interactions

The data indicate LAMTOR1 restricts lysosomal trafficking in dendrites through tonic inhibition of TRPML1 activity, establishing a novel regulatory mechanism for lysosomal positioning and function.

What methods can be used to validate LAMTOR1 antibody specificity in knockout/knockdown experiments?

To validate antibody specificity:

• Genetic validation approaches:

  • CRISPR/Cas9-mediated LAMTOR1 knockout

  • shRNA or siRNA-mediated knockdown (as demonstrated in the literature)

  • Rescue experiments with shRNA-resistant LAMTOR1 expression constructs

• Experimental validation workflow:

  • Generate LAMTOR1-depleted samples through knockdown or knockout

  • Compare antibody signal between control and LAMTOR1-depleted samples

  • Include rescue conditions to confirm specificity

  • Analyze by multiple detection methods (Western blot, immunofluorescence)

• Controls to include:

  • Positive control: Wild-type cells or tissues expressing LAMTOR1

  • Negative control: LAMTOR1 knockout cells

  • Specificity control: LAMTOR1 knockdown with rescue by shRNA-resistant constructs

  • Technical control: Secondary antibody only

• Quantification methods:

  • Densitometry analysis of Western blots

  • Fluorescence intensity measurements in immunofluorescence

  • Statistical comparison between experimental groups

In published studies, researchers validated LAMTOR1 knockdown efficiency by demonstrating loss of LAMTOR1 signal and restoration with rescue constructs , confirming antibody specificity.

How can researchers investigate LAMTOR1's interaction with the Ragulator complex and other protein partners?

To study LAMTOR1's protein interactions:

• Co-immunoprecipitation (Co-IP) approaches:

  • Use LAMTOR1 antibodies to pull down associated proteins

  • Alternatively, tag LAMTOR1 (Flag, YFP) for pull-down experiments

  • Analyze by Western blotting or mass spectrometry

• Structure-function studies:

  • Generate deletion constructs targeting specific domains:

    • N-terminal domain (residues 20-60)

    • C-terminal domain (residues 144-161)

    • Smaller deletions (e.g., residues 20-31 or 42-60)

  • Analyze their interaction capacity via Co-IP

• Proximity labeling techniques:

  • BioID or APEX2 fusions with LAMTOR1

  • TurboID for faster labeling kinetics

  • MS analysis of biotinylated proteins

• Fluorescence-based interaction studies:

  • FRET/BRET between LAMTOR1 and interaction partners

  • Fluorescence complementation assays

  • Live-cell imaging of co-localization

• In vitro binding assays:

  • Pull-down with recombinant proteins

  • Surface plasmon resonance

  • Isothermal titration calorimetry

Published data demonstrate successful use of co-IP techniques with tagged LAMTOR1 and TRPML1 constructs to study their interactions, revealing that the N-terminal domain of LAMTOR1 is critical for binding to TRPML1 .

What techniques can be employed to study LAMTOR1-dependent lysosomal trafficking and dynamics?

To investigate LAMTOR1's role in lysosomal trafficking:

• Live-cell imaging approaches:

  • LysoTracker labeling of acidic compartments

  • LAMP1-YFP expression to track lysosomes

  • Time-lapse microscopy to visualize trafficking

• Quantitative analysis methods:

  • Track lysosome movement (anterograde vs. retrograde)

  • Measure velocity and distance traveled

  • Analyze distribution patterns in cellular compartments

  • Quantify lysosome size and number

• Genetic manipulation strategies:

  • LAMTOR1 knockdown using shRNA or siRNA

  • Expression of domain-specific mutants

  • Rescue experiments with wild-type LAMTOR1

• Pharmacological interventions:

  • TRPML1 inhibitor (ML-SI1) to block downstream effects

  • TRPML1 activator (ML-SA1) to mimic LAMTOR1 depletion

  • GPN treatment to induce lysosomal osmotic lysis as control

• Experimental design considerations:

  • Cell-type specific effects (neurons vs. other cell types)

  • Compartment-specific analysis (dendrites vs. cell body)

  • Correlation with lysosomal properties (acidity, size)

Research has shown that LAMTOR1 knockdown increases the velocity and travel distance of lysosomes with higher fluorescent intensity (correlating with higher acidity), an effect that can be reversed by expressing shRNA-resistant LAMTOR1 .

How can researchers distinguish between mTORC1-dependent and independent functions of LAMTOR1?

Distinguishing between these functions requires careful experimental design:

• Comparative inhibition approaches:

  • LAMTOR1 knockdown/knockout vs. mTORC1 inhibition (rapamycin, Torin1)

  • Analysis of divergent phenotypes that indicate mTORC1-independent functions

• Domain-specific mutant analysis:

  • Generate LAMTOR1 mutants that selectively disrupt:

    • Interaction with Ragulator components (mTORC1-dependent)

    • Binding to TRPML1 or other partners (potentially mTORC1-independent)

  • Compare functional outcomes of each mutation

• Epistasis experiments:

  • Combined manipulation of LAMTOR1 and mTORC1

  • If phenotypes are additive, this suggests independent functions

  • If no additive effect, this suggests shared pathway

• Readout selection:

  • mTORC1-dependent processes: S6K phosphorylation, 4EBP1 phosphorylation

  • mTORC1-independent: TRPML1-mediated calcium release, lysosomal trafficking

• Temporal analysis:

  • Acute vs. chronic manipulation

  • Time-course studies to separate immediate vs. delayed effects

The research shows LAMTOR1 regulation of TRPML1-dependent lysosomal trafficking occurs independently of mTORC1, as demonstrated by specific inhibition of this pathway without affecting mTORC1 signaling .

What are common technical challenges when working with LAMTOR1 Antibody, HRP conjugated?

When using this antibody, researchers may encounter:

• Non-specific binding issues:

  • Optimize blocking conditions (concentration, type of blocking agent)

  • Adjust antibody dilution through titration experiments

  • Increase washing stringency (duration, buffer composition)

• Signal strength optimization:

  • Adjust substrate incubation time for HRP detection

  • Select appropriate substrate based on required sensitivity

  • Optimize sample protein concentration

• Detection limitations:

  • HRP conjugation may affect sensitivity in certain applications

  • Consider signal amplification systems for low-abundance targets

  • Ensure proper handling to prevent HRP activity loss

• Background reduction strategies:

  • Pre-adsorb antibody with non-specific proteins

  • Optimize incubation temperature and duration

  • Use detergents appropriate for your application

Each experimental system may require specific optimization to achieve optimal signal-to-noise ratio.

How can researchers standardize quantitative analyses of LAMTOR1 expression levels?

For reliable quantification:

• Sample normalization approaches:

  • Housekeeping protein normalization (β-actin, GAPDH, tubulin)

  • Total protein normalization (Ponceau, REVERT, Stain-Free technology)

  • Absolute quantification using recombinant protein standards

• Technical standardization:

  • Consistent protein extraction methods

  • Standard curve generation for each experiment

  • Use of biological and technical replicates

• Image acquisition standardization:

  • Consistent exposure settings

  • Linear dynamic range verification

  • Regular calibration of imaging equipment

• Data analysis considerations:

  • Background subtraction methods

  • Statistical approaches for comparing expression levels

  • Reporting relative vs. absolute expression levels

Standardized protocols ensure reproducibility and allow meaningful comparison between experimental conditions and across different studies.