LPHN3 Antibody

What is LPHN3 and what is its function in neural development?

LPHN3 (also known as ADGRL3) is a calcium-independent α-latrotoxin receptor that plays a crucial role in cell-cell adhesion and neuron guidance through its interactions with FLRT2 and FLRT3 expressed on adjacent cells . In neural development, LPHN3 is particularly important for determining connectivity rates between principal neurons in the cortex and plays a significant role in the development of glutamatergic synapses . The protein regulates synaptic function and maintenance in brain regions associated with locomotor activity, attention, and spatial memory processing . Research has demonstrated that LPHN3-FLRT complexes specifically regulate glutamatergic synaptic density in the CA1 region of the hippocampus where they modulate synaptic plasticity .

What are the key structural domains of LPHN3?

LPHN3 contains multiple functional domains that contribute to its role in synaptic development and function. While the search results don't provide a complete structural breakdown, we know that LPHN3 antibodies target specific regions including:

• N-terminal region (amino acids 371-397)

• C-terminal region

• Cytoplasmic domain

• Amino acids 500-650 within the human ADGRL3 protein

• Amino acids 1105-1447

This diverse targeting allows researchers to study different functional aspects of LPHN3 protein, including its extracellular interactions with binding partners and intracellular signaling mechanisms.

How does LPHN3 relate to neurodevelopmental disorders?

Variants of the LPHN3 gene are significantly associated with attention deficit hyperactivity disorder (ADHD) in multiple cohorts from different countries . These variants primarily occur in individuals with more severe ADHD symptoms of the combined type and can affect responses to psychostimulant medications . Specifically, polymorphisms in LPHN3 have been reported to increase the risk of ADHD by approximately 1.2-fold . Interestingly, there are no known null mutations of LPHN3 in humans, only variants that confer reduced protein expression and increased ADHD risk . Functional studies have revealed that LPHN3 variants are expressed in key brain regions related to attention and activity, affect metabolism in neural circuits implicated in ADHD, and are associated with response to stimulant medication .

What types of LPHN3 antibodies are available for research?

Based on the search results, several LPHN3 antibodies are available for research applications, primarily rabbit polyclonal antibodies targeting different regions of the protein:

These antibodies offer researchers flexibility in selecting the appropriate tool based on their experimental design, target species, and application requirements .

What applications are LPHN3 antibodies best suited for?

LPHN3 antibodies have been validated for several research applications according to the search results:

• Immunohistochemistry on paraffin-embedded tissue sections (IHC-P): Particularly useful for studying LPHN3 expression and localization in human tissue samples, as demonstrated in human stomach tissue where LPHN3 was detected in the membrane and cytoplasm of glandular cells .

• Western Blotting (WB): For protein expression analysis and quantification of LPHN3 levels in tissue or cell lysates .

• Immunoprecipitation (IP): To isolate LPHN3 protein complexes and study its interactions with binding partners such as FLRT2, FLRT3, and teneurins .

• Immunocytochemistry (ICC): For cellular localization studies, particularly useful when investigating the presynaptic versus postsynaptic localization of LPHN3 .

The optimal choice of application depends on the specific research question, with IHC and WB being the most commonly validated methods across available antibodies.

How can researchers validate the specificity of LPHN3 antibodies?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For LPHN3 antibodies, researchers should consider the following approaches:

• Positive and negative control tissues: Use tissues known to express or lack LPHN3, respectively. Human stomach tissue and uterine cervix tissue have been demonstrated to express LPHN3 and can serve as positive controls .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide (where available) to block specific binding sites.

• Multiple antibody approach: Use antibodies targeting different epitopes of LPHN3 to confirm observed expression patterns.

• Knockout/knockdown validation: Compare staining in wildtype versus LPHN3 knockout or knockdown samples. Some researchers have created LPHN3 knockout models that could serve as valuable negative controls .

• Immunogen sequence analysis: Verify that the antibody's immunogen sequence (e.g., aa 371-397 for ABIN1539334) is specific to LPHN3 and does not share homology with related proteins .

How can LPHN3 antibodies help investigate synaptic interactions?

LPHN3 forms trans-synaptic complexes with proteins like FLRT2/3 and teneurins to regulate synapse formation and function. Researchers can employ LPHN3 antibodies to study these interactions through:

• Co-immunoprecipitation: Using LPHN3 antibodies to pull down protein complexes, researchers can identify binding partners and study how these interactions change under different conditions or in disease models. This approach has been used to demonstrate LPHN3's interaction with FLRT proteins .

• Co-localization studies: Dual immunolabeling with LPHN3 antibodies and antibodies against potential binding partners (e.g., FLRT3) can reveal their spatial relationship at synapses. This technique has been valuable in investigating whether LPHN3 is predominantly presynaptic and FLRT3 is postsynaptic, though some questions remain about the specificity of available FLRT2 antibodies .

• Proximity ligation assays: This technique can detect protein-protein interactions between LPHN3 and its binding partners in situ with high sensitivity and specificity.

• Functional assays following antibody blockade: Using antibodies to block LPHN3's extracellular domain can help determine the functional significance of specific interactions in neuronal cultures.

These approaches have contributed to our understanding that LPHN3 and FLRT3 act as ligand-receptor modulators of excitatory synapses and that teneurins interact with LPHN3 during synapse formation .

What methodologies can be employed to study LPHN3's role in neural circuit development?

Researchers investigating LPHN3's role in neural circuit development can employ several methodologies:

• Developmental expression profiling: Using LPHN3 antibodies for immunohistochemistry or Western blotting across different developmental timepoints to map when and where LPHN3 expression is most critical.

• Synapse quantification: Combining LPHN3 immunolabeling with synaptic markers to quantify how LPHN3 affects synaptic density in specific brain regions, particularly in the cortex and hippocampus where LPHN3 has been shown to influence glutamatergic synapse development .

• Genetic manipulation studies: Using LPHN3 knockout models to study alterations in circuit connectivity, followed by rescue experiments with wildtype LPHN3. This approach revealed that LPHN3 acts trans-synaptically with adhesion molecules as ligands to provide specificity to synapse formation .

• Slice electrophysiology: Combining LPHN3 immunolabeling with functional recordings to correlate LPHN3 expression with synaptic physiology.

• In utero electroporation: To manipulate LPHN3 expression during critical developmental windows and subsequently study the effects on circuit formation.

These techniques have helped establish LPHN3's role in modulating excitatory synapses and determining connectivity patterns in the developing brain.

How might LPHN3 antibodies be used in ADHD research?

Given the established link between LPHN3 variants and ADHD, antibodies against this protein can be valuable tools in ADHD research:

• Comparative expression studies: Analyzing LPHN3 protein expression patterns in post-mortem brain tissue from individuals with and without ADHD-associated LPHN3 variants. Research has shown that certain variants confer reduced protein expression and increased ADHD risk .

• Pharmacological response studies: Examining how LPHN3 expression or localization changes in response to stimulant medications used to treat ADHD. This is particularly relevant since LPHN3 variants affect responses to ADHD medications .

• Animal model validation: Using LPHN3 antibodies to validate animal models of ADHD by confirming alterations in LPHN3 expression or localization that parallel human findings.

• Circuit-specific analyses: Investigating LPHN3 expression in brain circuits implicated in attention, impulse control, and hyperactivity - the core symptoms of ADHD.

• Biomarker development: Exploring whether LPHN3 protein levels or modifications could serve as biomarkers for ADHD subtypes or treatment response.

These approaches may help explain why LPHN3 variants are associated with more severe ADHD symptoms of the combined type and altered responses to ADHD medications .

What is the optimal protocol for LPHN3 immunohistochemistry?

Based on the search results, the following protocol has been successful for LPHN3 immunohistochemistry:

This protocol should be optimized based on the specific antibody, tissue type, and research question. Additional considerations like fixation time, section thickness, and blocking conditions may need adjustment for optimal results.

How can researchers troubleshoot weak or absent LPHN3 antibody staining?

When encountering weak or absent LPHN3 staining, researchers can try the following troubleshooting steps:

• Optimize antigen retrieval: The search results indicate heat-mediated antigen retrieval with citrate buffer (pH 6) works well , but alternative buffers or retrieval methods might be necessary depending on the fixation method and tissue type.

• Adjust antibody concentration: Test a range of antibody dilutions, potentially using a higher concentration than recommended initially (the 1/50 dilution used for ab150794 is relatively concentrated, suggesting LPHN3 may require higher antibody concentrations than many proteins) .

• Extend incubation time: Consider longer primary antibody incubation (overnight at 4°C).

• Check tissue quality and fixation: Overfixation can mask epitopes, while poor fixation can result in tissue degradation.

• Use amplification systems: Signal amplification methods (e.g., tyramide signal amplification) can enhance detection of low-abundance proteins.

• Consider epitope accessibility: Different LPHN3 antibodies target different regions of the protein, so if one antibody fails, try another targeting a different epitope .

• Verify LPHN3 expression: Confirm that the tissue being examined actually expresses LPHN3 using complementary methods like RT-PCR or Western blotting.

• Examine positive controls: Include tissues known to express LPHN3, such as human stomach or human uterine cervix .

What controls should be included in LPHN3 antibody experiments?

Proper controls are essential for interpreting LPHN3 antibody experiments:

• Positive tissue controls: Human stomach tissue and uterine cervix tissue have been validated for LPHN3 expression and can serve as positive controls .

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG for the antibodies in the search results)

  • Tissue known not to express LPHN3

  • Ideally, LPHN3 knockout tissue where available

• Peptide competition control: Pre-incubate the antibody with the immunizing peptide to block specific binding.

• Multiple antibody validation: When possible, verify findings using antibodies targeting different LPHN3 epitopes .

• Cross-reactivity controls: For studies in non-human species, verify the antibody's reactivity with the target species. Several LPHN3 antibodies react with human, mouse, and rat samples, but species-specific validation is still recommended .

These controls help ensure that observed staining is specific to LPHN3 and not due to non-specific binding or technical artifacts.

How can researchers quantify LPHN3 expression across different experimental conditions?

Quantifying LPHN3 expression can be approached through several methodologies:

• For Western blotting:

  • Normalize LPHN3 band intensity to loading controls (β-actin, GAPDH)

  • Use digital image analysis software to measure integrated density values

  • Apply statistical analyses appropriate for the experimental design (t-tests, ANOVA)

• For immunohistochemistry:

  • Measure staining intensity using color deconvolution and thresholding

  • Quantify percentage of LPHN3-positive cells in regions of interest

  • Assess subcellular localization patterns (membrane vs. cytoplasmic staining)

  • Use stereological approaches for unbiased quantification

• For immunofluorescence:

  • Measure fluorescence intensity in regions of interest

  • Analyze co-localization with synaptic markers

  • Perform high-content imaging for automated quantification

The specific approach should be consistent with the research question, such as whether LPHN3 localization changes in response to experimental manipulations or if expression levels differ between control and experimental groups.

How should contradicting results from different LPHN3 antibodies be interpreted?

When faced with contradicting results from different LPHN3 antibodies, researchers should consider several factors:

• Epitope differences: Different antibodies target different regions of LPHN3 (N-terminal, C-terminal, cytoplasmic domain) . These regions may have different accessibility depending on protein conformation, binding partners, or post-translational modifications.

• Isoform specificity: LPHN3 may have multiple isoforms, and different antibodies might recognize distinct isoforms.

• Methodological variables: Different antibodies may perform optimally under different conditions (fixation, antigen retrieval, buffer systems).

• Cross-reactivity: Some antibodies might cross-react with related proteins like LPHN1 or LPHN2.

To resolve contradictions:

• Perform multiple validation experiments (Western blot, IHC, ICC) using each antibody.

• Test in established positive and negative control tissues.

• Consider knockout/knockdown validation where available.

• Use orthogonal methods to verify findings (gene expression analysis, mass spectrometry).

• Consult literature for antibody validation data - as noted in the search results, some antibodies have better documented specificity than others .

The search results suggest that antibody specificity issues have affected FLRT2 research, highlighting the importance of rigorous validation .

What considerations are important when comparing LPHN3 expression data across species?

When comparing LPHN3 expression across species, researchers should consider:

• Epitope conservation: Verify that the epitope recognized by the antibody is conserved across the species being compared. The search results indicate that some LPHN3 antibodies react with multiple species (human, mouse, rat, chicken, horse, monkey), suggesting conservation of certain epitopes .

• Expression pattern validation: Even if an antibody recognizes LPHN3 across species, the expression pattern may differ. Validate findings with species-specific positive controls.

• Developmental timing differences: LPHN3 expression patterns may follow different developmental trajectories across species.

• Brain region homology: When comparing brain regions, consider anatomical differences and establish clear homologies between structures.

• Functional relevance: LPHN3's function in synaptic development appears conserved, but species-specific differences in LPHN3-related pathways may exist.

• Genetic variation: Consider that genetic variants associated with ADHD in humans may not have direct counterparts in other species .

These considerations help ensure that cross-species comparisons of LPHN3 expression are biologically meaningful and technically sound.