LRRTM4 Antibody, Biotin conjugated

What is LRRTM4 and what is its functional role in neural systems?

LRRTM4 (Leucine-rich repeat transmembrane neuronal protein 4) is a type I transmembrane protein that plays a crucial role in regulating glutamatergic synapse assembly on dendrites of central neurons. It contains 10 leucine-rich repeat (LRR) domains in its extracellular region and functions as a trans-synaptic adhesion protein .

In the mammalian brain, LRRTM4 shows region-specific expression with particularly high levels in the dentate gyrus of the hippocampus. In the retina, LRRTM4 is enriched at GABAergic synapses on axon terminals of rod bipolar cells (RBCs), where it regulates inhibitory synaptic organization . LRRTM4 knockout studies demonstrate reduced GABA receptor expression (both GABA-A and GABA-C receptors) on RBC terminals, indicating its importance for inhibitory synapse formation and maintenance .

What experimental applications are appropriate for biotin-conjugated LRRTM4 antibodies?

Biotin-conjugated LRRTM4 antibodies are suitable for multiple research applications:

• ELISA: Primary application with high sensitivity for protein detection

• Immunohistochemistry: Applicable for both paraffin-embedded (IHC-P) and frozen sections (IHC-F)

• Western Blot: When paired with streptavidin detection systems (typically at 1:1000 dilution)

• Expansion Microscopy: Certain LRRTM4 antibodies have been validated for magnified analysis of proteome (MAP) expansion microscopy methods (at 1:200 dilution)

The biotin conjugation provides significant signal amplification advantages through avidin/streptavidin interactions, making these antibodies particularly valuable for detecting low-abundance proteins using either the avidin-biotin complex (ABC) method or labeled streptavidin-biotin (LSAB) method .

What are the proper storage and handling requirements for biotin-conjugated LRRTM4 antibodies?

For optimal preservation of antibody functionality:

Reconstitution protocol:

• Add 50 μl H₂O to lyophilized antibody to achieve a 1mg/ml solution in PBS

• Allow complete dissolution

• Aliquot into small volumes to minimize freeze-thaw cycles

• Store aliquots at recommended temperatures

Critical handling notes:

• Avoid repeated freeze-thaw cycles as they significantly reduce antibody activity

• Some preparations contain stabilizers such as albumin, glycerol (50%), and preservatives like Proclin 300 (0.03%)

• Working solutions should be prepared fresh before experimental use

How does LRRTM4 antibody specificity vary across species and what validation steps are recommended?

Commercial LRRTM4 antibodies exhibit different species reactivity profiles:

Recommended validation approaches:

• Western blot analysis to confirm detection of bands at expected molecular weight (65-80 kDa)

• Include tissue from LRRTM4 knockout animals as negative controls when possible

• Compare staining patterns with known expression data from transcriptomic studies

• Use antibodies raised against different epitopes of LRRTM4 to confirm patterns

• Perform peptide competition assays to verify specificity

What is the significance of different immunogens used in LRRTM4 antibody production?

Different manufacturers produce antibodies targeting distinct regions of LRRTM4:

Experimental considerations:

• Antibodies targeting different epitopes may yield different staining patterns depending on protein conformation and accessibility

• Extracellular domain-targeting antibodies (N-terminal region) are typically suitable for cell surface detection in non-permeabilized conditions

• For detection of specific isoforms, consider epitope location relative to alternative splicing regions

• Recombinant protein immunogens may provide broader epitope recognition than peptide immunogens

How do LRRTM4 isoforms impact experimental design and antibody selection?

LRRTM4 exists in multiple isoforms that should be considered when designing experiments:

• Short isoform: Enriched in retina, cortex, and hippocampus relative to the long isoform

• Long isoform: Present in mouse and monkey retina but at lower levels

• Splice variants: Human LRRTM4 has at least one splice variant with Val substitution for cytoplasmic amino acids 518-590

Experimental design considerations:

• Verify which isoform is predominant in your tissue of interest

• Select antibodies whose epitopes are present in all isoforms of interest

• Use Western blotting to determine which isoforms are detected (bands at approximately 65 and 80 kDa have been reported)

• For developmental studies, consider potential changes in isoform expression across different stages

• When interpreting knockout phenotypes, confirm that all relevant isoforms are eliminated

What advantages do biotin-conjugated antibodies offer over unconjugated alternatives?

Biotin-conjugated LRRTM4 antibodies provide several technical advantages:

• Enhanced sensitivity: The biotin-avidin/streptavidin system amplifies signal due to:

  • High binding affinity (Kd=10⁻¹⁵ M) between biotin and avidin/streptavidin

  • Multiple biotin molecules conjugated to each antibody

  • Each avidin/streptavidin molecule binding up to four biotin molecules

• Detection flexibility: Compatibility with various detection systems:

  • Streptavidin-conjugated fluorophores for fluorescence microscopy

  • Enzyme-conjugated streptavidin for colorimetric/chemiluminescent detection

  • Gold-conjugated streptavidin for electron microscopy applications

• Signal amplification methods:

  • LSAB method: Typically provides higher specificity and better tissue penetration than ABC

  • ABC method: Uses preformed avidin-biotin complexes for detection

• Stability: The biotin-avidin interaction maintains stability under extreme conditions including various pH levels, temperatures, and exposure to organic solvents

What methodological modifications are necessary when using LRRTM4 antibodies for super-resolution microscopy?

Super-resolution microscopy with biotin-conjugated LRRTM4 antibodies requires specific technical considerations:

• Signal amplification optimization:

  • While biotin-streptavidin amplification increases signal, excessive amplification can compromise spatial resolution

  • For STED or STORM techniques, consider using monovalent streptavidin or directly labeled secondary antibodies for better resolution

  • Titrate concentration of both primary antibody and streptavidin-conjugate for optimal signal-to-noise ratio

• Sample preparation enhancements:

  • Consider expansion microscopy (ExM) - certain LRRTM4 antibodies have been validated for magnified analysis of proteome (MAP) methods (dilution 1:200)

  • Optimize fixation protocols to preserve antigenicity while maintaining synaptic ultrastructure

  • Reduce background by thorough blocking of endogenous biotin and careful washing steps

• Controls and validation:

  • Compare conventional confocal images with super-resolution data to ensure consistent localization patterns

  • Include known synaptic markers as reference points (e.g., synaptic ribbons labeled with CtBP2)

  • Consider correlative approaches with electron microscopy to validate findings

How should experimental design address LRRTM4's differential roles in excitatory versus inhibitory synapses?

LRRTM4 exhibits context-dependent functions at different synapse types:

• Experimental approach for excitatory synapses:

  • In hippocampal neurons, LRRTM4 regulates glutamatergic synapse assembly on dendrites

  • Co-label with markers of glutamatergic transmission (VGLUT, PSD-95)

  • Consider interactions with heparan sulfate proteoglycans (HSPGs) which mediate excitatory synapse development on dentate gyrus granule cells

• Approach for inhibitory synapses:

  • In retina, LRRTM4 is associated with GABAergic synapses on rod bipolar cell (RBC) axons

  • Co-label with GABA receptor markers (GABA-A α1, GABA-C)

  • Include electrophysiological measurements to distinguish between GABA-A and GABA-C-mediated responses

• Comparative analysis protocol:

  • Perform dual immunolabeling with both excitatory and inhibitory markers

  • Quantify colocalization coefficients

  • Analyze percent volume occupancy as a measure of receptor expression

  • Consider ribbon synapse markers (CtBP2) as control for synapse density in retina

What are the key considerations for triple immunolabeling experiments involving biotin-conjugated LRRTM4 antibodies?

Multiple labeling experiments with biotin-conjugated LRRTM4 antibodies require careful planning:

• Sequential labeling strategy:

  • Apply biotin-conjugated LRRTM4 antibody first

  • Detect with streptavidin-conjugated fluorophore

  • Block all unoccupied biotin-binding sites with excess biotin

  • Proceed with additional primary and secondary antibodies from different species

• Critical technical controls:

  • Single-labeled samples to establish baseline signals and assess spectral bleed-through

  • Biotin blocking controls to verify elimination of non-specific streptavidin binding

  • Secondary-only controls to detect non-specific binding

• Antibody compatibility matrix:

  • Select additional primary antibodies from host species different from the LRRTM4 antibody (typically rabbit)

  • Ensure secondary antibodies are highly cross-adsorbed to prevent cross-reactivity

  • Consider using directly conjugated antibodies for some targets to reduce complexity

• Signal separation strategies:

  • Select fluorophores with minimal spectral overlap

  • Optimize imaging settings with appropriate single-labeled controls

  • Consider spectral unmixing for closely overlapping fluorophores

  • Sequential scanning may be necessary to eliminate bleed-through in confocal applications

How can researchers troubleshoot unexpected results when using biotin-conjugated LRRTM4 antibodies?

What methodological approaches can distinguish LRRTM4 from other LRRTM family members?

The LRRTM family (LRRTM1-4) shares structural similarities that can complicate specific detection:

• Antibody validation protocol:

  • Test antibodies on overexpression systems with individual LRRTM family members

  • Verify absence of cross-reactivity with other family members in Western blot

  • Perform peptide competition assays with peptides from all family members

  • Compare immunostaining patterns with established expression patterns from in situ hybridization data

• Complementary approaches:

  • Combine protein detection with mRNA analysis using specific probes

  • Use RNAscope or similar methods for highly specific mRNA detection

  • Consider reporter gene knock-in models for unambiguous identification

• Knockout verification strategy:

  • Use tissue from LRRTM4 knockout mice as negative controls for antibody specificity

  • Check for compensatory expression of other LRRTM family members in knockout models

  • Evaluate potential redistribution of synaptic proteins in absence of LRRTM4

Research has shown that while LRRTM4 is restricted to certain brain regions with highest expression in the dentate gyrus, LRRTM1 and LRRTM2 show broader expression patterns , providing anatomical context for distinguishing between family members.