LPHN1 Antibody

What is LPHN1 and what are its key structural and functional features?

LPHN1 (Latrophilin-1), also known as ADGRL1, is a member of the adhesion G protein-coupled receptor subfamily. Like all GPCRs, Latrophilins have seven transmembrane domains and are distinguished by a large extracellular N-terminal tail and a large intracellular C-terminal tail . The N-terminus contains several cell adhesion domains and undergoes proteolysis after synthesis, while the C-terminal has various consensus post-translational sites like phosphorylation and palmitoylation .

Functionally, LPHN1 serves as a calcium-independent receptor with high affinity for α-latrotoxin, an excitatory neurotoxin from black widow spider venom that triggers massive exocytosis from neurons and neuroendocrine cells . Recent studies have identified LPHN1 as a receptor for teneurin-2 (TENM2) that mediates heterophilic synaptic cell-cell contact and postsynaptic specialization . Notably, LPHN1 plays a crucial role in inhibitory synapse formation, particularly those near the neuronal soma .

What applications are LPHN1 antibodies suitable for?

LPHN1 antibodies are validated for multiple experimental applications:

When planning experiments, it's important to note that certain antibodies perform better in specific applications. For example, the extracellular antibodies are particularly useful for cell surface detection in live, intact cells, as demonstrated with Anti-LPHN1 (extracellular) antibody in cell surface detection of Latrophilin-1 by indirect flow cytometry in live intact human THP-1 monocytic leukemia cells .

What species reactivity do commonly used LPHN1 antibodies show?

Various LPHN1 antibodies display cross-reactivity across multiple species, which is important when designing comparative studies:

Some antibodies show particularly broad cross-reactivity. For instance, BLAST analysis of the peptide immunogen for the Anti-LPHN1 (C-Term) antibody shows 100% identity with Human, Gorilla, Gibbon, Monkey, Marmoset, Mouse, Rat, Hamster, Elephant, and Platypus, 94% identity with Bat, Bovine, Horse, Pig, and Opossum, and 88% identity with Dog and Panda .

What are the different epitope targets available for LPHN1 antibodies?

LPHN1 antibodies targeting different regions of the protein are available, each with specific advantages for different experimental questions:

• N-Terminal/Extracellular Antibodies:

  • Anti-LPHN1 (extracellular) Antibody (#ALR-021): Targets amino acid residues 480-494 (peptide CEPREVRRVQWPATQ) of rat Latrophilin-1, located in the extracellular N-terminus .

  • Anti-LPHN1 antibody (N-terminal) (ab140825): Targets the N-terminal region of human ADGRL1 .

• C-Terminal Antibodies:

  • Anti-LPHN1 (C-Term) antibody (ABIN1049023): Targets the C-terminus of human LPHN1 .

• Internal Region Antibodies:

  • Anti-LPHN1 antibody (Abbexa): The antiserum was produced against a synthesized peptide derived from the internal region of human LPHN1 .

  • Anti-LPHN1 ADGRL1 Antibody (A30832): Targets a peptide derived from human LPHN1, immunogen sequence location 561-610 .

For experiments involving live cells where membrane-bound LPHN1 needs to be detected without cell permeabilization, extracellular epitope-targeting antibodies are particularly useful .

How can LPHN1 antibodies be used to study synaptic nanoclusters?

Recent research has revealed that LPHN1 forms nanoclusters in both excitatory and inhibitory synapses of hippocampal neurons . When investigating these nanoclusters, consider the following methodological approach:

• Super-resolution microscopy: STED (Stimulated Emission Depletion) microscopy has been successfully used to visualize LPHN1 nanoclusters in synapses . This technique provides the necessary resolution to distinguish individual clusters that conventional confocal microscopy cannot resolve.

• Epitope tagging approach: Studies have utilized myc-tagged LPHN1 in conditional knockout mice to overcome the limitations of antibody specificity. In these systems, an extracellular myc epitope tag is inserted into the endogenous LPHN1 protein, allowing highly specific detection with anti-myc antibodies .

• Quantitative analysis of nanoclusters: When analyzing LPHN1 nanoclusters, researchers have quantified:

  • Number of nanoclusters per synapse (found to be higher in somatic inhibitory synapses with ~2.4 nanoclusters compared to dendritic inhibitory synapses with ~1.8 nanoclusters and excitatory synapses with ~1.3 nanoclusters) .

  • Distribution patterns in different synaptic types (excitatory vs. inhibitory).

• Co-labeling strategies: Combine LPHN1 antibody labeling with markers for excitatory (e.g., PSD-95) and inhibitory (e.g., gephyrin) synapses to analyze differential distribution .

This approach has revealed that LPHN1 nanoclusters are more abundant in inhibitory synapses than in excitatory synapses, particularly in somatic inhibitory synapses .

What considerations are important when studying LPHN1's role in both excitatory and inhibitory synapses?

When investigating LPHN1's differential roles in excitatory versus inhibitory synapses, researchers should consider:

• Synapse-specific localization patterns: LPHN1 forms nanoclusters in both excitatory and inhibitory synapses but shows preferential functional importance for inhibitory synapses, particularly those near the neuronal soma . This contrasts with Lphn2 and Lphn3, which primarily impact excitatory synapses.

• Conditional knockout approaches: Conditional deletion of LPHN1 in cultured neurons failed to elicit detectable impairment in excitatory synapses but produced a decrease in inhibitory synapse numbers and synaptic transmission that was most pronounced for synapses close to the neuronal soma .

• Quantitative analysis methodology: When evaluating synaptic parameters after LPHN1 manipulation, measure:

  • Synapse numbers (density)

  • Pre- and postsynaptic areas (which may show small, statistically insignificant trends to being smaller in LPHN1-deficient neurons)

  • Synapse-marker staining intensities

  • Electrophysiological parameters of synaptic transmission

• Controls for conditional systems: When using Cre-mediated deletion in conditional knockout systems, include proper controls such as ΔCre virus-infected cultures that should maintain normal LPHN1 expression (showing ~115 kDa bands in immunoblots) versus Cre virus-infected cultures showing >97% reduction of LPHN1 protein expression .

These considerations will help distinguish LPHN1's unique contributions to different synapse types from those of other latrophilin family members.

How can researchers distinguish between different latrophilin isoforms in their experiments?

Distinguishing between the three latrophilin isoforms (LPHN1, LPHN2, LPHN3) is crucial for understanding their specific functions. Recommended approaches include:

Using a combination of these approaches provides the most reliable isoform distinction, especially important when studying brain regions where multiple latrophilins are expressed.

How can LPHN1 antibodies be used to investigate the protein's role in obesity and metabolic disorders?

Recent research has identified LPHN1 as a potential factor in metabolic regulation, with implications for obesity development:

• Tissue-specific expression analysis: Use immunohistochemistry with LPHN1 antibodies to characterize expression patterns in metabolically relevant tissues. Studies have shown that Lphn1 knockout mice develop obesity over time by accumulating excess fat, with significant changes in liver morphology showing fat accumulation .

• Analysis of metabolic pathways: Investigate LPHN1's impact on:

  • Glucose tolerance and insulin sensitivity, which are decreased in Lphn1 deficient mice

  • Lipolysis regulation, as male Lphn1 knockout mice exhibit reduced non-esterified fatty acids (NEFA) levels compared to wild-type controls

  • Lipase expression in adipose tissue, with significant increases in ΔCt values of hormone-sensitive lipase (Hsl) and lipoprotein lipase (Lpl) observed in male animals lacking LPHN1

• Sex-specific differences: Design experiments accounting for sex differences in LPHN1's metabolic effects. In Lphn1 knockout mice, certain effects on lipolysis were observed only in males but not females , suggesting hormonal interactions.

• Correlation with human pathologies: A partially inactivating mutation in human ADGRL1/LPHN1 has been identified in a patient suffering from obesity , suggesting translational relevance of animal model findings.

• Signaling pathway analysis: Investigate LPHN1's potential impact on protein kinase A (PKA) phosphorylation, which controls the activity of many lipases and thus lipolysis. Some studies observed a tendency toward reduced phosphorylation in subcutaneous white adipose tissue of male Lphn1 knockout mice .

This emerging research area suggests LPHN1 as a novel target for understanding metabolic disorders beyond its well-established neuronal functions.

What are the optimal sample preparation protocols for different LPHN1 antibody applications?

Effective sample preparation is crucial for successful LPHN1 detection across different applications:

For Immunohistochemistry (IHC):

• Fixation: Formalin fixation followed by paraffin embedding is recommended .

• Sectioning: Make 4-μm sections and place on pre-cleaned and charged microscope slides .

• Heat treatment: Heat slides in a tissue-drying oven for 45 minutes at 60°C .

• Deparaffinization: Wash slides in 3 changes of xylene (5 minutes each) at room temperature .

• Rehydration: Use a graded alcohol series (3 changes of 100% alcohol for 3 minutes each, 2 changes of 95% alcohol for 3 minutes each, 1 change of 80% alcohol for 3 minutes) .

• Antigen retrieval: Steam slides in 0.01 M sodium citrate buffer, pH 6.0 at 99-100°C for 20 minutes .

For Live Cell Surface Labeling:

• Use extracellular epitope-targeting antibodies such as Anti-LPHN1 (extracellular) antibody (#ALR-021) .

• For flow cytometry: Use approximately 2.5μg antibody per sample followed by fluorescently-labeled secondary antibody (e.g., goat-anti-rabbit-FITC) .

• For fluorescence microscopy of live cells: Dilute primary antibody (e.g., 1:50 for Anti-LPHN1 extracellular antibody) in appropriate buffer and apply to live, non-permeabilized cells followed by fluorophore-conjugated secondary antibody (e.g., goat anti-rabbit-AlexaFluor-594) .

For Western Blot:

• For brain tissue samples: Prepare lysates from brain regions of interest, with cerebral cortex and hippocampus showing strong LPHN1 expression .

• Include appropriate positive controls (tissues or cells known to express LPHN1) and negative controls (LPHN1 knockout tissues where available) .

• Be aware that LPHN1 undergoes autoproteolytic cleavage, resulting in an N-terminal fragment of approximately 115 kDa , which may be the predominant band detected with N-terminal targeting antibodies.

What dilutions are typically recommended for different applications of LPHN1 antibodies?

Optimal antibody dilutions vary by application and specific antibody:

These recommendations provide starting points for optimization. Researchers should perform dilution series to determine optimal concentrations for their specific experimental conditions and sample types.

What controls are essential when using LPHN1 antibodies, particularly in knockout/conditional knockout studies?

Rigorous controls are crucial for ensuring valid results with LPHN1 antibodies:

• Positive controls:

  • Known LPHN1-expressing tissues/cells (e.g., brain lysates, neuronal cultures)

  • Recombinant LPHN1 protein where available

• Negative controls:

  • Primary antibody omission

  • LPHN1 knockout or knockdown samples

  • In conditional knockout systems, compare Cre virus-infected cultures (showing >97% reduction of LPHN1) versus ΔCre virus-infected cultures (maintaining normal LPHN1 expression)

• Specificity controls:

  • Preincubation with blocking peptide: For example, Anti-LPHN1 (extracellular) Antibody preincubated with Latrophilin-1/LPHN1 (extracellular) Blocking Peptide (#BLP-LR021)

  • Multiple antibodies targeting different epitopes should yield consistent results

  • BLAST analysis of immunogen sequences to assess potential cross-reactivity

• Technical controls:

  • For immunofluorescence: Include nuclear counterstain (e.g., DAPI) to aid in cell identification

  • For Western blot: Include loading controls and molecular weight markers (expected LPHN1 N-terminal fragment at ~115 kDa following autoproteolysis)

  • For quantitative analyses: Include standardization controls across different experimental batches

• Validation approaches:

  • Orthogonal methods (combining protein detection with mRNA analysis)

  • Epitope tagging in genetic models (e.g., myc-tagged LPHN1 in conditional knockout mice)

These controls are particularly important given the reported challenges with LPHN1 antibody specificity, as mentioned in several studies .

What are common challenges when working with LPHN1 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with LPHN1 antibodies:

• Antibody specificity issues:

  • Challenge: Multiple studies note that "no highly specific LPHN1 antibodies are available" .

  • Solution: Use genetic approaches such as epitope tagging in knockin mice , utilize knockout controls, and validate with multiple antibodies targeting different epitopes.

• Detection of multiple bands in Western blots:

  • Challenge: LPHN1 undergoes autoproteolytic cleavage by its GAIN domain, resulting in different-sized fragments .

  • Solution: Be aware that N-terminal antibodies may detect primarily the ~115 kDa N-terminal fragment rather than the full-length protein . C-terminal antibodies will detect different fragments.

• Species cross-reactivity limitations:

  • Challenge: Some antibodies show limited cross-species reactivity.

  • Solution: Carefully review the species reactivity information for each antibody and validate with species-specific positive controls.

• Background in immunohistochemistry/immunofluorescence:

  • Challenge: High background can obscure specific LPHN1 signals.

  • Solution: Optimize blocking conditions (typically 5-10% serum from the species of the secondary antibody), increase washing steps, and titrate primary antibody dilutions. For brain tissue, consider autofluorescence quenching steps.

• Detection in live cells:

  • Challenge: Maintaining cell viability while achieving good signal.

  • Solution: Use extracellular domain-targeting antibodies specifically designed for live cell applications , minimize incubation times, and conduct procedures at 4°C to reduce internalization.

• Heterogeneous expression patterns:

  • Challenge: LPHN1 expression can vary across different cell types and brain regions.

  • Solution: Include appropriate positive controls and analyze multiple fields/sections to account for heterogeneity.

These solutions should help researchers overcome common technical hurdles when working with LPHN1 antibodies.

How can contradictory results between different LPHN1 antibodies be resolved?

When faced with contradictory results between different LPHN1 antibodies, consider the following systematic approach:

• Epitope analysis:

  • Different antibodies target different regions of LPHN1 (N-terminal/extracellular, C-terminal, internal) .

  • Contradictory results may reflect differential accessibility of epitopes or detection of different protein fragments following post-translational modifications.

  • Review whether the targeted epitopes might be affected by known LPHN1 processing events, such as autoproteolytic cleavage by the GAIN domain .

• Validation with genetic approaches:

  • Use LPHN1 knockout or knockdown models as definitive controls .

  • Consider using epitope-tagged LPHN1 knockin models, such as myc-tagged LPHN1 , which allow detection with highly specific anti-tag antibodies.

• Cross-validation with orthogonal methods:

  • Combine immunodetection with mRNA analysis (qPCR, in situ hybridization).

  • Perform mass spectrometry identification of immunoprecipitated proteins.

  • Use functional assays based on known LPHN1 activities, such as α-latrotoxin binding or interaction with teneurin-2 .

• Technical standardization:

  • Ensure all antibodies are tested under identical conditions (same samples, preparation methods, detection systems).

  • Perform side-by-side comparisons with detailed documentation of all experimental variables.

• Antibody characterization:

  • Review the validation data for each antibody, including knockout controls and preabsorption tests.

  • Consider generating your own validation data using LPHN1-overexpressing cells versus control cells.

• Reconciliation of divergent findings:

  • Different antibodies may reveal distinct aspects of LPHN1 biology, such as differential localization of processed fragments.

  • Document the specific conditions under which each antibody works optimally and the specific LPHN1 form or state it detects.

This systematic approach should help resolve apparent contradictions and may even reveal new insights into LPHN1 biology.