LRRTM1 Antibody

What is LRRTM1 and why is it significant for neuroscience research?

LRRTM1 (leucine-rich repeat transmembrane neuronal 1) is a 522 amino acid single-pass type I membrane protein predominantly localized to the endoplasmic reticulum. The protein features ten leucine-rich repeats forming a hydrophobic α/β horseshoe fold critical for structural integrity and protein-protein interactions . LRRTM1 is primarily expressed in forebrain tissue where it plays significant roles in neuronal differentiation, connectivity, and axon trafficking during development . The high conservation of LRRTM1 across species (human LRRTM1 shares 96% amino acid identity with mouse LRRTM1) suggests evolutionary importance of its function . Defects in the gene encoding LRRTM1 have been implicated in several neurodevelopmental disorders, making it a significant target for researchers investigating brain development, synaptic function, and neurological conditions .

How does LRRTM1 contribute to synaptic development and function?

LRRTM1 contributes to synaptic development through its role as a synaptogenic adhesion molecule. LRRTM1 participates in excitatory synapse formation and maintenance, particularly in forebrain structures . The protein facilitates proper axon trafficking and neuronal connectivity during development through its leucine-rich repeat domains that mediate protein-protein interactions . Studies using LRRTM1/2 double knockout mice have demonstrated altered synaptic density and morphology, confirming its critical role in synaptogenesis . LRRTM1 also appears to influence synaptic convergence in visual thalamus, indicating its role extends to sensory processing circuits . The protein likely works in concert with other synaptic adhesion molecules, with LRRTM1 and LRRTM2 showing distinct but overlapping roles in synapse development . These functions make LRRTM1 essential for proper brain wiring and subsequent cognitive function.

What types of LRRTM1 antibodies are currently available for research applications?

Multiple LRRTM1 antibodies suitable for various research applications are currently available. These include:

The monoclonal antibody from Santa Cruz is available in multiple formats including non-conjugated, agarose-conjugated, HRP-conjugated, and fluorophore-conjugated versions (PE, FITC, Alexa Fluor) . This variety allows researchers to select the most appropriate antibody format for specific experimental needs, whether for protein detection, localization studies, or quantitative analyses . The use of different host species also provides flexibility when designing multiplex experiments requiring co-staining with other antibodies.

What is the recommended protocol for brain tissue immunostaining with LRRTM1 antibodies?

The recommended protocol for LRRTM1 immunostaining in brain tissue, based on published methodologies, includes:

• Tissue Preparation:

  • Anesthetize animals (e.g., with 20% urethane or isoflurane)

  • Perform transcardial perfusion with cold 0.1M PBS followed by 4% formaldehyde/4% sucrose in PBS (pH 7.4)

  • Post-fix brains overnight in 4% formaldehyde

  • Cryoprotect in sequential 20% and 30% sucrose solutions in PBS at 4°C

  • Freeze in OCT compound and section to 20μm thickness using a cryostat

• Immunostaining:

  • Wash sections with TBSTr (50mM Tris pH 7.4, 1.5% NaCl, 0.3% TritonX-100) for 20 minutes

  • Incubate in blocking solution (TBSTr containing 10% normal goat serum) for 1 hour

  • Apply primary LRRTM1 antibody (e.g., rabbit anti-LRRTM1, Alomone Labs) overnight at 4°C

  • Wash with TBSTr for 1 hour

  • Apply appropriate secondary antibodies conjugated to fluorophores (e.g., Alexa 568) for 1.5 hours at room temperature

  • Wash with TBSTr for 20 minutes followed by 50mM Tris (pH 7.4) for 30 minutes

  • Mount sections using appropriate medium containing DAPI

• Controls and Co-staining:

  • Include LRRTM1 knockout tissue as negative controls when available

  • For synaptic studies, co-stain with markers such as VGlut1 (excitatory presynaptic) or GAD65 (inhibitory presynaptic)

  • Acquire images under consistent microscope settings for quantitative analyses

How should LRRTM1 antibodies be validated before use in critical experiments?

Comprehensive validation of LRRTM1 antibodies should include:

• Specificity Testing:

  • Western blot analysis using positive control samples (e.g., SH-SY5Y neuroblastoma cells) to confirm detection of expected ~65kDa band

  • Testing in tissue/cells from LRRTM1 knockout or knockdown models as negative controls

  • Peptide competition assays where pre-incubation of antibody with excess target peptide should abolish specific signal

• Cross-Reactivity Assessment:

  • Testing against closely related proteins (other LRRTM family members)

  • Verifying species reactivity claims by testing in multiple species (human, mouse, rat)

  • Confirming absence of non-specific binding to other proteins in Western blots

• Application-Specific Validation:

  • For immunohistochemistry: comparing staining patterns with published literature

  • For Western blotting: confirming band size and testing multiple sample types

  • For immunoprecipitation: verifying pull-down efficiency and specificity

• Reproducibility Testing:

  • Testing antibody performance across multiple lots

  • Ensuring consistent results across different experimental conditions

  • Comparing results with alternative antibodies targeting different LRRTM1 epitopes

This systematic validation approach increases confidence in experimental outcomes and helps prevent misleading results due to antibody limitations or batch variations.

What is the optimal sample preparation method for detecting LRRTM1 in Western blots?

The optimal sample preparation method for detecting LRRTM1 in Western blots includes:

• Lysate Preparation:

  • For cell lines (e.g., SH-SY5Y): harvest cells at 80-90% confluence

  • For brain tissue: rapidly dissect and flash-freeze tissue before homogenization

  • Use lysis buffer containing appropriate detergents and protease inhibitors

  • Incubate on ice for 30 minutes with occasional vortexing

  • Centrifuge at 14,000×g for 15 minutes at 4°C to remove debris

  • Determine protein concentration using standard assay (BCA or Bradford)

• Sample Processing:

  • Mix samples with reducing sample buffer (containing DTT or β-mercaptoethanol)

  • Heat at 95°C for 5 minutes to ensure complete denaturation

  • Load 20-50μg total protein per lane for cell/tissue lysates

• Electrophoresis Parameters:

  • Use 8-10% SDS-PAGE gels for optimal resolution of LRRTM1 (~65kDa)

  • Include molecular weight markers covering 50-75kDa range

  • Run at constant voltage (e.g., 100V) until sufficient separation is achieved

• Transfer Conditions:

  • Transfer to PVDF membrane (preferred over nitrocellulose for LRRTM1)

  • Use wet transfer system at 100V for 1 hour or 30V overnight at 4°C

  • Verify transfer efficiency with reversible protein stain before blocking

• Immunoblotting:

  • Block with 5% non-fat dry milk or BSA in TBST

  • Incubate with LRRTM1 antibody at recommended dilution (e.g., 1μg/mL for R&D Systems AF4897)

  • Use appropriate HRP-conjugated secondary antibody

  • Visualize using enhanced chemiluminescence detection

This protocol has been validated to detect LRRTM1 as a specific band at approximately 65kDa in human neuroblastoma cell lysates .

How can researchers differentiate between LRRTM family members in experimental systems?

Differentiating between LRRTM family members requires specific strategies:

• Antibody Selection:

  • Use antibodies raised against unique epitopes that differ between LRRTM1, LRRTM2, LRRTM3, and LRRTM4

  • Validate specificity through Western blotting using recombinant proteins for each family member

  • Consider using antibodies from different host species to enable co-staining approaches

• Experimental Approaches:

  • Multiplex immunofluorescence: Simultaneously detect multiple LRRTM family members using antibodies with different fluorophores

  • Sequential immunoprecipitation: Selectively deplete one family member before analyzing others

  • Western blot analysis: Compare molecular weights (LRRTM1 at ~65kDa) and expression patterns across tissues

• Genetic Controls:

  • Use tissue from single, double, or triple knockout animals (e.g., LRRTM1/2-DKO as described in source )

  • Employ siRNA or shRNA knockdown of specific LRRTM family members

  • Create overexpression systems with tagged versions of each family member

• Expression Pattern Analysis:

  • Compare regional distribution patterns (LRRTM1 is primarily expressed in forebrain)

  • Analyze cell-type specificity of expression

  • Correlate with in situ hybridization data for different LRRTM mRNAs

A successful example from the literature includes differential staining for LRRTM1 (using rabbit antibody from Alomone Labs) and LRRTM2 (using antibody 510KSCN) in brain sections, enabling researchers to identify distinct and overlapping roles of these family members in synapse development .

What approaches can be used to study LRRTM1's role in synapse formation and function?

Multiple complementary approaches can be employed to investigate LRRTM1's role in synapse formation:

• Genetic Manipulation Models:

  • Constitutive or conditional LRRTM1 knockout mice

  • LRRTM1/2 double knockout models to account for compensatory mechanisms

  • Viral-mediated knockdown or overexpression of LRRTM1 in specific brain regions

  • Rescue experiments with wild-type or mutant LRRTM1 constructs

• Structural Analysis:

  • Electron microscopy to quantify synapse density, PSD length, and synaptic vesicle distribution

  • Immunofluorescence co-staining with pre- and post-synaptic markers (VGlut1, GAD65)

  • Super-resolution microscopy to examine nanoscale organization of synaptic proteins

  • Live imaging of fluorescently tagged LRRTM1 to track dynamics during synaptogenesis

• Functional Assessment:

  • Electrophysiological recordings (mEPSCs, mIPSCs, evoked responses)

  • Calcium imaging to assess synaptic activity patterns

  • Synaptic vesicle recycling assays using FM dyes or pHluorin-based reporters

  • Behavioral assays to assess circuit-level functional consequences

• Molecular Interaction Studies:

  • Co-immunoprecipitation with LRRTM1 antibodies to identify binding partners

  • Proximity ligation assays to visualize protein-protein interactions in situ

  • Proteomic analysis of synapse composition in wild-type vs. LRRTM1 knockout models

These approaches have revealed LRRTM1's importance in excitatory synapse development and its role in synaptic convergence in visual thalamus , providing a framework for investigating this protein's function in normal development and disease states.

How can researchers design experiments to analyze the effect of LRRTM1 mutations on protein function?

Designing experiments to analyze LRRTM1 mutations requires a multifaceted approach:

• Mutation Selection and Generation:

  • Focus on mutations in functionally important domains (leucine-rich repeats, transmembrane domain)

  • Consider naturally occurring mutations identified in human studies

  • Generate mutations using site-directed mutagenesis in expression constructs

  • Create knock-in mouse models with specific mutations using CRISPR/Cas9

• Protein Expression and Localization:

  • Compare expression levels of wild-type and mutant LRRTM1 using Western blotting

  • Analyze subcellular localization using immunofluorescence with LRRTM1 antibodies

  • Assess surface expression using biotinylation assays or surface immunostaining

  • Evaluate protein stability and turnover rates using pulse-chase experiments

• Molecular Interaction Studies:

  • Test binding of mutant LRRTM1 to known interaction partners using co-immunoprecipitation

  • Perform binding assays with recombinant proteins to quantify affinity changes

  • Use yeast two-hybrid or mammalian two-hybrid assays for interaction screening

  • Analyze protein complex formation using blue native PAGE

• Functional Consequences:

  • Compare synaptogenic activity of wild-type vs. mutant LRRTM1 in co-culture assays

  • Perform rescue experiments in LRRTM1 knockout neurons

  • Analyze synaptic density and morphology using electron microscopy

  • Assess electrophysiological properties in neurons expressing mutant LRRTM1

• In Vivo Relevance:

  • Generate knock-in mouse models harboring specific mutations

  • Analyze brain development, synaptic connectivity, and behavior

  • Perform circuit-specific functional analyses using electrophysiology or imaging

  • Correlate findings with human data when available

This systematic approach enables researchers to establish clear genotype-phenotype relationships and understand how specific LRRTM1 mutations might contribute to neurodevelopmental disorders.

What are common troubleshooting approaches for non-specific binding of LRRTM1 antibodies?

When encountering non-specific binding with LRRTM1 antibodies, consider these troubleshooting strategies:

• Blocking Optimization:

  • Increase blocking reagent concentration (5-10% normal serum from secondary antibody species)

  • Add additional blocking agents (1-5% BSA, 0.1-0.3% Triton X-100)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Use commercial blocking solutions specifically designed to reduce background

• Antibody Dilution and Incubation:

  • Test a range of primary antibody dilutions to find optimal signal-to-noise ratio

  • Reduce antibody concentration if background is high

  • Increase incubation time while decreasing antibody concentration

  • Perform antibody incubations at 4°C instead of room temperature

• Washing Procedures:

  • Increase number and duration of washing steps

  • Use TBSTr buffer (50mM Tris pH 7.4, 1.5% NaCl, 0.3% TritonX-100) as described in successful protocols

  • Ensure thorough washing between primary and secondary antibody incubations

• Controls and Validation:

  • Include tissue from LRRTM1 knockout animals as negative controls

  • Perform secondary-only controls to identify secondary antibody background

  • Use peptide competition assays to confirm specificity

  • Test multiple antibodies against different epitopes of LRRTM1

• Sample-Specific Approaches:

  • For brain tissue with high lipid content, consider adding additional permeabilization steps

  • Pre-adsorb antibodies with tissue homogenates from knockout animals

  • For fluorescence applications, use Sudan Black B (0.1-0.3%) to reduce lipofuscin autofluorescence

These approaches have successfully enabled specific LRRTM1 detection in brain tissue using appropriate antibodies and protocols .

How should researchers interpret apparent molecular weight differences of LRRTM1 in Western blot experiments?

Interpreting molecular weight variations of LRRTM1 in Western blots requires consideration of several factors:

• Expected Molecular Weight Profile:

  • The predicted molecular weight of human LRRTM1 based on amino acid sequence is approximately 59kDa

  • LRRTM1 typically migrates at approximately 65kDa on SDS-PAGE under reducing conditions

  • This discrepancy often reflects post-translational modifications

• Post-translational Modifications:

  • LRRTM1 undergoes N-glycosylation, which can add 5-10kDa to the apparent molecular weight

  • Treatment with glycosidases (PNGase F) can confirm glycosylation status

  • Phosphorylation may result in additional molecular weight shifts

• Sample-Specific Variations:

  • Different species may show slight variations in LRRTM1 migration patterns

  • Tissue-specific or developmental differences in post-translational modifications

  • Cell-type specific processing or protein complex formation

• Technical Considerations:

  • Different gel systems (gradient vs. fixed percentage) affect migration patterns

  • Buffer conditions and reducing agent concentrations influence protein mobility

  • Sample preparation methods (heating temperature, time) may cause variability

• Interpretation Guidelines:

  • Always include molecular weight markers and positive controls

  • Compare observed bands with literature reports (e.g., the 65kDa band reported for human LRRTM1)

  • Verify specificity using knockout tissues/cells or peptide competition

  • Consider running samples on different percentage gels to confirm identity

Understanding these factors helps researchers correctly identify LRRTM1 bands and interpret variations that might reflect biologically relevant modifications rather than artifacts.

What strategies can be employed to quantitatively compare LRRTM1 expression across different brain regions?

Quantitative comparison of LRRTM1 expression across brain regions requires:

• Immunohistochemistry-Based Quantification:

  • Use standardized immunostaining protocols with validated LRRTM1 antibodies

  • Process all tissues simultaneously under identical conditions

  • Acquire images using consistent microscope settings (exposure, gain, offset)

  • Analyze using automated image analysis software to measure:

    • Staining intensity (integrated density or mean gray value)

    • Area of expression

    • Cell counts (for cellular expression patterns)

  • Normalize to established housekeeping proteins or total protein content

• Western Blot Quantification:

  • Dissect discrete brain regions precisely

  • Extract proteins using consistent protocols

  • Load equal amounts of total protein from each region

  • Include recombinant LRRTM1 standards for absolute quantification

  • Use fluorescence-based detection for wider linear range

  • Normalize to stable reference proteins (β-actin, GAPDH)

• Complementary Approaches:

  • qRT-PCR for mRNA expression analysis to correlate with protein levels

  • In situ hybridization to provide cellular resolution of expression patterns

  • Mass spectrometry-based proteomics for absolute quantification

  • Single-cell techniques to identify cell type-specific expression profiles

• Statistical Considerations:

  • Use appropriate statistical tests for multiple region comparisons (ANOVA with post-hoc tests)

  • Include sufficient biological replicates (minimum n=3-6 animals per group)

  • Consider age, sex, and strain as potential variables

  • Report effect sizes alongside p-values

These approaches have successfully revealed region-specific expression patterns of LRRTM1, particularly its enrichment in forebrain regions and involvement in specific circuits such as the visual thalamus .

How can researchers ensure reproducibility when comparing results from different LRRTM1 antibodies?

Ensuring reproducibility when comparing different LRRTM1 antibodies requires:

• Antibody Characterization:

  • Document epitope information for each antibody used

  • Determine antibody type (monoclonal vs. polyclonal) and host species

  • Record manufacturer, catalog number, and lot number

  • Report working dilutions and optimization procedures

• Validation Strategy:

  • Validate each antibody independently using knockout/knockdown controls

  • Perform peptide competition assays for each antibody

  • Include appropriate positive controls for each antibody

  • Test for cross-reactivity with other LRRTM family members

• Experimental Design:

  • Process samples in parallel using standardized protocols

  • Include internal controls in each experiment

  • Blind analysis to prevent bias

  • Replicate experiments multiple times with different antibody lots

• Comparative Analysis:

  • Directly compare staining patterns in adjacent sections

  • Quantify correlation between signals from different antibodies

  • Document concordant and discordant findings

  • Consider that discrepancies might reveal biologically relevant information

• Reporting Standards:

  • Follow ARRIVE guidelines for reporting animal research

  • Document detailed methods including all antibody information

  • Report both positive and negative results

  • Share raw data and analysis methods when possible

This approach enhances reliability and facilitates comparison of results across different laboratories, contributing to more robust findings in LRRTM1 research.

How might new technologies advance our understanding of LRRTM1 function in neural circuits?

Emerging technologies offer exciting opportunities for LRRTM1 research:

• Advanced Imaging Approaches:

  • Super-resolution microscopy (STORM, STED) to visualize LRRTM1 nanoscale organization at synapses

  • Expansion microscopy to physically enlarge specimens for improved resolution

  • Lattice light-sheet microscopy for high-speed volumetric imaging of LRRTM1 dynamics

  • Cryo-electron tomography to reveal LRRTM1 in its native environment at molecular resolution

• Genetic Engineering Tools:

  • CRISPR/Cas9-based genomic editing for precise manipulation of LRRTM1

  • Split protein complementation assays to visualize LRRTM1 interactions in living neurons

  • Inducible expression systems for temporal control of LRRTM1 function

  • Cell type-specific manipulation using intersectional genetic approaches

• Functional Analysis Technologies:

  • Optogenetics combined with LRRTM1 manipulation to assess circuit-level consequences

  • Calcium imaging in behaving animals to correlate LRRTM1 function with neural activity

  • Connectomics approaches to map LRRTM1-dependent synaptic networks

  • Fiber photometry to monitor long-term activity in LRRTM1-expressing circuits

• Proteomics and Structural Biology:

  • Proximity labeling approaches (BioID, APEX) to identify the LRRTM1 interactome in vivo

  • Hydrogen-deuterium exchange mass spectrometry to map LRRTM1 interaction surfaces

  • Cryo-EM structural analysis of LRRTM1 in complex with binding partners

  • AlphaFold2 predictions to guide structure-function studies of LRRTM1

These technologies, combined with validated LRRTM1 antibodies, will advance our understanding of this protein's role in neural circuit development and function.

What are promising research directions for understanding LRRTM1's role in neurodevelopmental disorders?

Several promising research directions for LRRTM1 in neurodevelopmental disorders include:

• Human Genetic Studies:

  • Whole genome/exome sequencing to identify LRRTM1 variants in patient cohorts

  • Case-control studies examining LRRTM1 variants in specific disorders

  • Functional characterization of disease-associated variants

  • Population genetics approaches to understand LRRTM1 variation across human groups

• Patient-Derived Models:

  • iPSC-derived neurons from patients with LRRTM1 mutations

  • Organoid models to study LRRTM1 function in 3D developing neural tissue

  • CRISPR-engineered isogenic lines to isolate effects of specific mutations

  • Transcriptomic and proteomic profiling of patient-derived models

• Circuit-Level Analysis:

  • Investigation of LRRTM1's role in specific circuits implicated in neurodevelopmental disorders

  • Analysis of LRRTM1 expression in post-mortem brain tissue from patients

  • Studies examining interaction between LRRTM1 and other risk genes

  • Identification of critical developmental windows when LRRTM1 function is most crucial

• Therapeutic Development:

  • Screening for compounds that modulate LRRTM1 function or expression

  • Development of antibody-based approaches to target LRRTM1 signaling

  • Gene therapy approaches to correct LRRTM1 deficiency

  • Circuit-specific interventions targeting LRRTM1-dependent synapses

These research directions build upon current knowledge of LRRTM1's role in forebrain development and synaptic function , potentially leading to new insights into neurodevelopmental disorders and novel therapeutic approaches.