LRTM1 Antibody

What is LRTM1 protein and why is it relevant to neuroscience research?

LRTM1 is a leucine-rich repeat membrane protein that plays a crucial role in the positive regulation of synapse assembly and functions as an integral membrane component. It has highest expression in the pineal body and is involved in synaptic plasticity and neurodevelopment . The protein's role in learning and memory formation makes it particularly relevant to neuroscience research, as dysregulation of LRTM1 has been implicated in various neurological disorders including autism and schizophrenia . Understanding LRTM1's function can provide insights into normal brain development and potential therapeutic targets for neurological conditions.

What are the validated applications for LRTM1 antibodies in experimental research?

LRTM1 antibodies have been validated for multiple experimental applications:

• Western Blot: Detecting LRTM1 at approximately 75 kDa under reducing conditions

• Flow Cytometry: Identifying LRTM1-expressing cells using APC-conjugated secondary antibodies

• Immunohistochemistry (IHC): Detecting LRTM1 in fixed paraffin-embedded tissue sections

• Immunocytochemistry (ICC): Visualizing LRTM1 in fixed cell lines

• Immunofluorescence (IF): Localizing LRTM1 in cellular compartments

• ELISA: Quantitative detection of LRTM1 protein

What cell and tissue types have shown positive LRTM1 expression?

Research has demonstrated positive LRTM1 expression in:

• TK-6 human lymphoblastoid cell line (positive cytoplasmic staining)

• HEK293T cells when transfected with human LRTM1

• Human spleen tissue (cytoplasmic localization)

• Pineal body (highest expression level)

Notably, A549 human lung carcinoma cell line shows negative staining for LRTM1, making it a potential negative control for experiments .

How should optimal dilutions for LRTM1 antibodies be determined across different applications?

Determining optimal antibody dilutions requires systematic testing for each application and experimental condition. Based on manufacturer recommendations and validated protocols:

Each laboratory should establish a dilution series to determine optimal signal-to-noise ratio for their specific experimental conditions .

What are the optimal storage and handling conditions for maintaining LRTM1 antibody functionality?

To preserve antibody functionality and minimize loss of activity:

• Long-term storage: Keep at -20°C to -70°C for up to 12 months from receipt date

• Medium-term storage: Store at 2-8°C under sterile conditions for up to 1 month after reconstitution

• Extended storage after reconstitution: Store at -20°C to -70°C for up to 6 months under sterile conditions

• Avoid repeated freeze-thaw cycles by aliquoting upon initial thaw

• Storage buffer considerations: Most LRTM1 antibodies are stored in preservative buffers (e.g., 0.03% Proclin 300) with 50% glycerol and 0.01M PBS at pH 7.4

What controls should be included when validating LRTM1 antibody specificity?

A robust experimental design should include these controls:

• Positive controls:

  • HEK293T cells transfected with human LRTM1 (validated in western blot and flow cytometry)

  • TK-6 human lymphoblastoid cell line (positive for endogenous expression)

• Negative controls:

  • Mock-transfected HEK293T cells

  • A549 human lung carcinoma cells (consistently negative for LRTM1)

  • Irrelevant transfectants paired with eGFP for flow cytometry

  • Isotype control antibodies (e.g., MAB1050) for setting quadrant markers in flow cytometry

• Technical controls:

  • Secondary antibody-only staining to assess non-specific binding

  • Validation across multiple lots when possible

  • Cross-verification with alternative detection methods

How can LRTM1 antibodies be utilized to investigate synaptic plasticity mechanisms?

Investigating LRTM1's role in synaptic plasticity requires sophisticated experimental approaches:

• Co-localization studies: Use LRTM1 antibodies in combination with synaptic markers (PSD-95, synaptophysin) to examine spatial relationships at excitatory synapses through super-resolution microscopy or confocal imaging.

• Proximity ligation assays: Employ LRTM1 antibodies with antibodies against potential binding partners to visualize and quantify protein-protein interactions at synapses with single-molecule resolution.

• Synaptosome preparations: Isolate synaptosomes and probe for LRTM1 enrichment using the antibody to determine subcellular localization during different phases of synaptic plasticity.

• Activity-dependent changes: Monitor LRTM1 expression and localization following paradigms that induce long-term potentiation (LTP) or long-term depression (LTD) to correlate protein dynamics with functional changes .

• Receptor complex immunoprecipitation: Use LRTM1 antibodies for pull-down experiments to identify novel interacting proteins within the synaptic machinery, potentially using a membrane protein display platform similar to that described for other membrane proteins .

What methodological considerations are important when investigating LRTM1 in neurological disorder models?

Researchers studying LRTM1 in neurological disorder contexts should consider:

• Model selection considerations:

  • Patient-derived iPSCs differentiated into neurons for disease-relevant LRTM1 expression patterns

  • Animal models with conditionally regulated LRTM1 expression to study developmental timing effects

  • Brain region-specific analysis focusing on areas with high LRTM1 expression or relevance to the disorder

• Technical approaches:

  • Quantitative immunohistochemistry with standardized image analysis protocols

  • Single-cell protein quantification using flow cytometry with LRTM1 antibodies

  • Multiplexed protein detection combining LRTM1 with other neurological disorder-associated markers

• Developmental timeline analysis:

  • Track LRTM1 expression throughout neurodevelopment in control versus disease models

  • Correlate expression patterns with emergence of phenotypic characteristics

  • Implement inducible knockdown/overexpression systems to determine critical periods

• Functional correlation:

  • Pair LRTM1 antibody-based detection with electrophysiological recordings to correlate protein levels with functional synaptic measures

  • Implement long-term imaging protocols to track LRTM1 dynamics during circuit formation

How can researchers address potential cross-reactivity when studying LRTM family proteins?

The LRTM protein family shares structural similarities that may complicate specific detection. Researchers should:

• Implement epitope mapping:

  • Determine the specific epitope recognized by the antibody

  • Verify epitope conservation or divergence across LRTM family members

  • Design blocking peptides specific to the epitope region for validation experiments

• Perform cross-validation:

  • Use multiple LRTM1 antibodies targeting different epitopes

  • Compare monoclonal (e.g., MAB10046) versus polyclonal (e.g., PACO41954) antibody results

  • Validate with genetic approaches (siRNA knockdown, CRISPR knockout)

• Conduct specificity assays:

  • Test antibody reactivity against recombinant LRTM family proteins

  • Perform pre-absorption controls with purified LRTM1 and related family members

  • Include heterologous expression systems with individual LRTM family members

• Employ bioinformatic analysis:

  • Conduct sequence alignment to identify unique regions suitable for specific detection

  • Predict potential cross-reactive epitopes based on structural similarities

  • Design custom antibodies targeting LRTM1-specific regions if commercial options show cross-reactivity

What are common causes of false negative results when using LRTM1 antibodies?

False negative results may stem from several methodological issues:

• Sample preparation problems:

  • Inadequate epitope exposure: LRTM1 detection in tissue sections requires heat-induced epitope retrieval using basic antigen retrieval reagents (e.g., CTS013)

  • Inappropriate fixation: Overfixation can mask epitopes, particularly for membrane proteins like LRTM1

  • Buffer incompatibility: Using incorrect immunoblot buffer groups can reduce detection sensitivity (Immunoblot Buffer Group 1 is recommended)

• Technical factors:

  • Insufficient antibody concentration: LRTM1 may require higher concentrations (2-3 μg/mL) than typical for optimal detection

  • Inappropriate detection systems: Signal amplification may be necessary for low-abundance expression

  • Suboptimal incubation conditions: Some applications require extended incubation (3 hours at room temperature)

• Biological considerations:

  • Cell type-dependent expression: LRTM1 shows negative staining in A549 cells but positive in TK-6 cells

  • Expression level variations: LRTM1 has highest expression in pineal body but may be below detection threshold in other tissues

  • Post-translational modifications: These may mask epitopes in certain cellular contexts

How should researchers interpret disparities between LRTM1 detection methods?

When facing inconsistent results across detection methods:

• Protein form considerations:

  • Western blot detects denatured LRTM1 at approximately 75 kDa, while flow cytometry and ICC detect native conformations

  • Post-translational modifications may differ across cell types affecting epitope accessibility

  • Protein complexes or interactions may mask epitopes in certain applications

• Methodological sensitivity hierarchy:

  • ELISA (1:2000-1:10000 dilution) generally provides highest sensitivity

  • Western blot (1:1000-1:5000 dilution) offers good sensitivity for denatured protein

  • IHC/ICC/IF (1:20-1:200) have moderate sensitivity dependent on expression level

• Systematic validation approach:

  • Confirm antibody specificity using positive controls (HEK293T transfected cells) and negative controls (A549 cells)

  • Perform parallel detection with alternative antibodies targeting different epitopes

  • Complement protein detection with mRNA analysis (qPCR, RNA-seq) to corroborate expression patterns

What strategies can address membrane protein-specific challenges when working with LRTM1 antibodies?

LRTM1, as an integral membrane protein, presents unique experimental challenges:

• Enhanced solubilization strategies:

  • Optimize lysis buffers with appropriate detergents (mild non-ionic for native conformation, stronger ionic detergents for complete solubilization)

  • Consider using membrane protein extraction kits specifically designed for transmembrane proteins

  • Implement ultrasonication or mechanical disruption to improve membrane protein extraction

• Membrane protein display platforms:

  • Consider using recombinant extracellular vesicles (rEVs) for displaying LRTM1 in its native membrane environment

  • HIV gag-containing vesicles can provide a platform for studying LRTM1 interactions

  • This approach can enhance detection of membrane-dependent interactions that may not be visible with solubilized protein

• Advanced detection methods:

  • Apply proximity ligation assays for detecting LRTM1 interactions within membrane microdomains

  • Consider label-free detection methods that bypass potential interference with protein function

  • Implement biolayer interferometry (BLI) for studying LRTM1 binding kinetics in membrane contexts

How might LRTM1 antibodies contribute to understanding receptor-ligand interactions in neurological contexts?

LRTM1 antibodies could facilitate several innovative research approaches:

• Receptor-ligand discovery:

  • Implement receptor deorphanization studies using LRTM1 antibodies in combination with candidate screening approaches

  • Adapt the RDIMIS (Receptor Deorphanization by Integral Membrane Interaction Screening) platform described for other membrane proteins to identify LRTM1 binding partners

  • Develop blocking antibodies to functionally characterize newly identified interactions

• Synaptic specificity analysis:

  • Map LRTM1 expression across synapse types using multi-label immunohistochemistry

  • Correlate LRTM1 levels with specific circuit functions in defined neuronal populations

  • Track developmental regulation of LRTM1-mediated interactions during circuit formation

• Therapeutic target validation:

  • Use LRTM1 antibodies to assess target engagement in preclinical models

  • Develop function-blocking antibodies to modulate LRTM1 activity in neurological disorder models

  • Implement tissue-specific analysis of LRTM1 expression in patient samples to identify disease-relevant alterations

What methodological advances might improve LRTM1 detection in complex tissue samples?

Emerging technologies could enhance LRTM1 detection specificity and sensitivity:

• Multiplexed imaging approaches:

  • Cyclic immunofluorescence to correlate LRTM1 with multiple markers in the same sample

  • Mass cytometry with metal-conjugated LRTM1 antibodies for high-dimensional analysis

  • Spatial transcriptomics combined with LRTM1 protein detection for correlating protein localization with gene expression patterns

• Enhanced sensitivity methods:

  • Signal amplification systems (tyramide signal amplification, rolling circle amplification)

  • Quantum dot-conjugated antibodies for improved signal-to-noise ratio

  • Super-resolution microscopy techniques optimized for membrane protein visualization

• Single-cell protein analysis:

  • Flow cytometry panels incorporating LRTM1 with other neurological markers

  • Mass cytometry for high-parameter analysis of LRTM1 in heterogeneous neural populations

  • Single-cell western blot approaches for quantifying LRTM1 in individual cells

How can researchers leverage LRTM1 antibodies to investigate extracellular vesicle-mediated signaling?

The study of LRTM1 in extracellular vesicle contexts represents an emerging frontier:

• EV isolation and characterization:

  • Use LRTM1 antibodies to immunoprecipitate specific subpopulations of EVs

  • Develop flow cytometry approaches for LRTM1+ vesicle quantification

  • Implement density gradient fractionation followed by LRTM1 immunoblotting to characterize vesicle subtypes

• Functional studies:

  • Track LRTM1+ vesicle uptake using fluorescently labeled antibodies

  • Correlate LRTM1 presentation on vesicles with recipient cell responses

  • Develop vesicle display platforms similar to those described for other membrane proteins to study LRTM1 function

• Biomarker development:

  • Assess LRTM1+ vesicles in biofluids from patients with neurological disorders

  • Develop sensitive detection methods for LRTM1 on circulating vesicles

  • Correlate LRTM1+ vesicle signatures with disease progression or treatment response