LRRTM4 Antibody

What is LRRTM4 and why is it significant in neuroscience research?

LRRTM4 is a 67.2 kilodalton transmembrane glycoprotein belonging to the LRRTM family. It plays a crucial role in the development and maintenance of the vertebrate nervous system, particularly in excitatory presynaptic differentiation . Found predominantly in neurons of the cerebral cortex, dentate gyrus granule cells, and cerebellar Purkinje cells, LRRTM4 contains 10 external leucine-rich repeats (LRRs) and a cytoplasmic tail that binds PDF motifs . Its involvement in synaptic function makes it an important target for neurodevelopmental and neurological disorder research.

How should I select the appropriate LRRTM4 antibody for my specific experiment?

When selecting an LRRTM4 antibody, consider:

• Target region specificity: Determine whether you need antibodies targeting the N-terminal, middle region, or C-terminal domains. For instance, antibodies targeting the middle region (aa 259-419) offer access to the LRR domains crucial for protein-protein interactions .

• Application compatibility: Verify the antibody's validated applications match your experimental needs:

  • Western blotting typically requires denatured epitope recognition

  • Immunohistochemistry requires preservation of epitopes through fixation processes

  • Immunofluorescence requires specific fluorophore compatibility

• Species reactivity: Ensure cross-reactivity with your experimental model (human, mouse, rat, etc.) .

• Clonality: Polyclonal antibodies provide broader epitope recognition but potential batch variation, while monoclonal and recombinant antibodies offer higher specificity and reproducibility .

What are the optimal conditions for Western blot detection of LRRTM4?

For optimal Western blot detection of LRRTM4:

• Sample preparation: Use brain tissue lysates where LRRTM4 is highly expressed. PVDF membrane is recommended for protein transfer .

• Antibody concentration: Start with 1 μg/mL concentration for primary antibody incubation, as demonstrated in successful detections using sheep anti-human LRRTM4 antibodies .

• Expected band pattern: Prepare to observe specific bands at approximately 65 kDa and 80 kDa, which represent different glycosylation states of LRRTM4 .

• Buffer conditions: Use reducing conditions with appropriate immunoblot buffer groups (e.g., Immunoblot Buffer Group 8 has been successfully used) .

• Secondary antibody selection: Choose species-appropriate HRP-conjugated secondary antibodies for optimal detection .

How can I optimize immunohistochemical detection of LRRTM4 in brain tissue sections?

Optimizing IHC detection of LRRTM4 in brain tissue requires:

• Tissue fixation: Use paraformaldehyde fixation to preserve protein structure while maintaining tissue architecture.

• Antigen retrieval: Apply heat-induced epitope retrieval in citrate buffer (pH 6.0) to expose LRRTM4 epitopes masked during fixation.

• Antibody selection: Choose antibodies specifically validated for IHC-P (paraffin) or IHC-fro (frozen) applications, depending on your tissue preparation .

• Signal localization: Expect signal primarily in neuronal cell bodies and dendrites in the cerebral cortex, dentate gyrus, and cerebellar Purkinje cells .

• Control validation: Include both positive controls (known LRRTM4-expressing tissues) and negative controls (primary antibody omission or blocking peptide competition) .

How can I address non-specific binding when using LRRTM4 antibodies?

To reduce non-specific binding:

• Blocking optimization: Use 5% non-fat milk or BSA in TBS-T for Western blotting; for immunostaining, include 10% serum from the same species as the secondary antibody.

• Validation with blocking peptides: Employ LRRTM4 blocking peptides (e.g., catalog no. 33R-3143) alongside the primary antibody to confirm signal specificity .

• Antibody titration: Test multiple dilutions to determine the optimal concentration that maximizes specific signal while minimizing background.

• Cross-adsorption: Select antibodies that have been cross-adsorbed against related proteins to reduce cross-reactivity with other LRRTM family members.

• Buffer modifications: Adjust salt concentration and detergent levels to optimize the signal-to-noise ratio.

What controls should be included when validating a new LRRTM4 antibody?

Essential controls include:

• Positive tissue controls: Human brain tissue lysates for Western blotting or tissue sections for IHC/IF, as LRRTM4 is known to be expressed in cerebral cortex, dentate gyrus, and cerebellar Purkinje cells .

• Negative controls:

  • Primary antibody omission

  • Pre-immune serum substitution

  • Competitive blocking with immunizing peptide

• Knockdown/knockout validation: If available, tissues or cells with LRRTM4 gene silencing or deletion provide the most stringent specificity control.

• Recombinant protein: Western blot with recombinant LRRTM4 protein can establish size specificity.

• Comparison with alternative antibodies: Using antibodies targeting different LRRTM4 epitopes provides orthogonal validation.

How can LRRTM4 antibodies be used to investigate synaptic development in neuronal cultures?

For synaptic development studies:

• Co-localization analysis: Use LRRTM4 antibodies alongside presynaptic (e.g., Bassoon, Synaptophysin) and postsynaptic (e.g., PSD95) markers in immunofluorescence to quantify synaptic connections .

• Live-cell imaging: Apply membrane-impermeable antibodies against the extracellular domain of LRRTM4 to track surface expression in living neurons.

• Proximity ligation assay (PLA): Combine LRRTM4 antibodies with antibodies against potential binding partners to detect protein-protein interactions with nanometer resolution.

• Functional perturbation: Apply function-blocking LRRTM4 antibodies to neuronal cultures to assess the consequences of disrupting LRRTM4 interactions on synapse formation.

• Time-course analysis: Utilize LRRTM4 antibodies at different developmental timepoints to track expression changes during synaptogenesis.

What are the challenges in detecting LRRTM4 in non-CNS tissues and how can they be overcome?

Challenges and solutions for detecting LRRTM4 in non-CNS tissues:

• Low expression levels: Use signal amplification methods such as tyramide signal amplification or high-sensitivity detection systems.

• Background interference: Apply antigen retrieval optimization specific to each tissue type and extended blocking steps.

• Epitope accessibility: Test multiple antibodies targeting different LRRTM4 regions to identify the most accessible epitopes in different tissue contexts .

• Cross-reactivity concerns: Employ parallel detection with different antibodies targeting the same protein to confirm specificity.

• Fixation artifacts: Compare multiple fixation protocols to determine optimal epitope preservation in non-neural tissues.

How do LRRTM4 expression patterns compare across different species, and which antibodies are optimal for cross-species studies?

For cross-species LRRTM4 studies:

• Sequence homology: Human LRRTM4 shares 95% amino acid identity with mouse LRRTM4 over amino acids 31-424, making this region ideal for cross-species antibody targeting .

• Recommended antibodies: Select antibodies validated for multiple species reactivity (human, mouse, rat) with epitopes in conserved regions .

• Species differences:

  Species	Key Expression Sites	Optimal Antibody Applications
  Human	Cerebral cortex, dentate gyrus, cerebellar Purkinje cells	WB, IHC, IF
  Mouse	Similar to human, with slight variations in hippocampal expression	WB, IHC, IF
  Rat	Similar to human and mouse, useful for detailed dendritic studies	WB, IHC

• Validation strategy: Perform parallel experiments in tissue from multiple species to confirm antibody performance across models.

What methods are available for studying the glycosylation state of LRRTM4 and how can antibodies facilitate this research?

To study LRRTM4 glycosylation:

• Band pattern analysis: LRRTM4 appears as multiple bands (65 kDa and 80 kDa) in Western blots due to different glycosylation states .

• Enzymatic deglycosylation: Treat samples with PNGase F or Endoglycosidase H before immunoblotting to distinguish N-linked glycosylation patterns.

• Glycosylation-specific antibodies: Some antibodies may preferentially detect specific glycoforms; compare multiple antibodies targeting different epitopes.

• Lectin co-staining: Combine LRRTM4 antibody labeling with specific lectins to characterize glycan structures.

• Mutational analysis: Use antibodies to compare glycosylation patterns between wild-type LRRTM4 and site-directed mutants of potential glycosylation sites.

How can LRRTM4 antibodies be utilized in studies of neurodevelopmental disorders?

Applications in neurodevelopmental disorder research:

• Post-mortem tissue analysis: Compare LRRTM4 expression and localization in tissue from individuals with autism, schizophrenia, or intellectual disabilities versus controls.

• Animal model validation: Use LRRTM4 antibodies to assess protein expression in genetic models of neurodevelopmental disorders.

• Synapse quantification: Employ LRRTM4 antibodies alongside synaptic markers to evaluate excitatory synapse numbers and morphology in disorder models.

• Circuit-specific analysis: Combine LRRTM4 immunostaining with circuit tracers to assess pathway-specific alterations in disorder models.

• Therapeutic screening: Use LRRTM4 immunoassays to evaluate candidate therapeutics' effects on synaptic development.

What are the most effective strategies for studying LRRTM4's role in transsynaptic signaling using antibody-based approaches?

Strategies for transsynaptic signaling studies:

• Co-immunoprecipitation: Use LRRTM4 antibodies to pull down protein complexes, followed by mass spectrometry to identify binding partners .

• Super-resolution microscopy: Apply LRRTM4 antibodies in STORM or STED microscopy to visualize nanoscale organization at synapses.

• Synaptosome preparations: Fractionate brain tissue and use LRRTM4 antibodies to track protein distribution across synaptic compartments.

• Activity-dependent changes: Monitor LRRTM4 localization changes following neuronal activity using antibody labeling of fixed samples at various timepoints.

• Receptor complex formation: Use bifunctional crosslinking approaches combined with LRRTM4 immunoprecipitation to capture transient interaction complexes at the synapse.