LPHN2 Antibody

What is LPHN2 and what are its key biological functions?

LPHN2 is an adhesion G protein-coupled receptor that plays multiple roles in different tissues:

• In cochlear hair cells, LPHN2 is expressed at the tips of stereocilia and is associated with mechanoelectrical transduction (MET) channel components, acting as a force-sensing GPCR essential for normal auditory function

• In the hippocampus, LPHN2 mediates precise neural circuit assembly through interactions with partners like teneurin-3, functioning as both a repulsive receptor in distal CA1 neurons and as a repulsive ligand in the proximal subiculum

• Recent studies suggest LPHN2 may serve as a receptor for LRG1 (leucine-rich α-2-glycoprotein 1), potentially promoting angiogenic and neurotrophic processes under hyperglycemic conditions

LPHN2 has molecular weight of approximately 163-220 kDa, undergoes autoproteolytic cleavage, and contains multiple domains including RBL, OLF, HRM, and GAIN domains, plus a seven transmembrane helix region .

What applications are LPHN2 antibodies commonly used for in research?

Based on commercially available antibodies and published studies, LPHN2 antibodies are primarily used for:

• Immunohistochemistry (IHC) on paraffin-embedded tissues (1:50-1:100 dilution)

• Immunofluorescence (IF) for cellular localization studies (1:100-1:500 dilution)

• Western blotting (WB) to detect protein expression (1:500-1:1000 dilution)

• ELISA for quantitative detection (typically 1:1000 dilution)

• Flow cytometry for cell surface or intracellular detection

These applications have been successfully employed to study LPHN2 in neural tissues, cochlear hair cells, and various other cell types.

What epitopes are commonly targeted by research-grade LPHN2 antibodies?

LPHN2 antibodies target different regions of the protein:

The N-terminal region (amino acids 551-600) is particularly significant as it's been used to develop antibodies capable of exerting quantifiable force on LPHN2 in a magnetic field for functional studies .

How can I validate the specificity of my LPHN2 antibody?

Proper validation is crucial for ensuring experimental rigor:

• Genetic approaches: Use LPHN2 knockout tissues (Lphn2-/- cochleae) or knockdown cells as negative controls

• Overexpression systems: Test antibody in HEK293 cells overexpressing LPHN2 versus empty vector controls

• Peptide competition: Pre-incubate antibody with the immunizing peptide before application to samples

• Cross-reactivity assessment: Test on tissues known to express versus not express LPHN2 (e.g., LPHN2 is found in cochlear hair cell stereocilia but not in utricular hair cell stereocilia)

• Western blot molecular weight verification: Confirm the detection of bands at expected sizes (~110-220 kDa depending on glycosylation and processing state)

For example, researchers have validated anti-LPHN2 antibodies by demonstrating specific staining in LPHN2-expressing HEK293 cells that was absent in mock-transfected cells, confirming both specificity and appropriate subcellular localization .

What are the key considerations for using LPHN2 antibodies in subcellular localization studies?

When examining LPHN2's subcellular distribution:

• Separation techniques: Consider using a modified twist-off method to separate stereocilia from cell bodies when studying LPHN2 in hair cells

• Co-localization markers: Use TMIE/PCDH15 as markers for stereocilia and Myosin7a for cell bodies in cochlear preparations

• Optical sectioning: Collect different optical sections ranging from stereocilia to cell body to properly examine localization patterns

• Fixation protocol: Use 4% paraformaldehyde for most tissue preparations, but optimize fixation time based on tissue type

• Permeabilization method: For intracellular epitopes, use Flow Cytometry Permeabilization/Wash Buffer or similar reagents that maintain epitope integrity

Research has shown that LPHN2 localizes differently across tissues - for example, it's found in both stereocilia and cell bodies of cochlear hair cells but only in the cell body of utricular hair cells .

How can I use LPHN2 antibodies to study G protein coupling and signaling pathways?

For signaling studies:

• Force application experiments: Coat paramagnetic beads with anti-LPHN2 antibodies (LPHN2-M-beads) to exert quantifiable force on LPHN2 in a magnetic field

• Pathway activation measurement: After force application, measure cAMP levels to assess Gs and Gi signaling responses

• Mutant analysis: Compare signaling between wild-type LPHN2 and constructs with mutations in the tethered agonist region or autoproteolytic cleavage site

• Downstream effector detection: Monitor PI3K, AKT, and NF-κB p65 activation as key components of the LPHN2 signaling pathway

Research has demonstrated that force application to LPHN2 via antibody-coated beads induces a dose-dependent increase in cAMP concentration in LPHN2-expressing cells but not in control cells transfected with empty vector .

What is the recommended protocol for LPHN2 immunohistochemistry in neural tissues?

For optimal IHC results in neural tissues:

• Tissue preparation:

  • Fix tissue in 4% paraformaldehyde

  • Process and embed in paraffin

  • Section at 4-6 μm thickness

• Antigen retrieval:

  • Perform heat-induced antigen retrieval (specific buffer may vary by antibody)

• Blocking and antibody incubation:

  • Block with 10% normal serum in TBS-T for 1 hour at room temperature

  • Incubate with primary anti-LPHN2 antibody (typically 10-14 μg/ml) overnight at 4°C

  • Wash 3× with TBS-T

  • Incubate with appropriate HRP-conjugated secondary antibody for 2 hours at room temperature

  • Develop with DAB substrate and counterstain as needed

• Controls:

  • Include primary antibody omission control

  • When possible, include peptide competition control

  • Include positive control tissue with known LPHN2 expression (e.g., prostate tissue)

This protocol has been successfully used to detect LPHN2 in various tissues including vessel walls, prostate, and brain sections .

How should I optimize Western blot protocols for LPHN2 detection?

For effective Western blot detection:

• Sample preparation:

  • Extract proteins using standard lysis buffers containing protease inhibitors

  • Given LPHN2's large size (163-220 kDa), use lower percentage gels (6-8%) or gradient gels

• Electrophoresis and transfer:

  • Extend transfer time for large proteins (overnight at lower voltage is often effective)

  • Consider using PVDF membrane rather than nitrocellulose for better retention of high MW proteins

• Antibody incubation:

  • Block membrane with 5% non-fat dry milk or BSA in TBS-T for 1 hour

  • Incubate with anti-LPHN2 antibody (typically 1:500-1:1000 dilution) overnight at 4°C

  • Wash thoroughly with TBS-T (3× for 10 minutes each)

  • Incubate with HRP-conjugated secondary antibody for 1-2 hours at room temperature

  • Detect using ECL substrate and appropriate imaging method

• Controls and interpretation:

  • Include positive control (LPHN2-transfected cell lysate)

  • Be aware that LPHN2 undergoes proteolytic processing, resulting in multiple bands

  • Full-length LPHN2 appears at ~163-220 kDa while cleaved C-terminal fragment appears at ~72 kDa

For quantitative analysis, researchers have used ImageJ software for densitometry analysis of scanned Western blot films .

What is the optimal procedure for using LPHN2 antibodies in flow cytometry?

For flow cytometry applications:

• Cell preparation:

  • For surface staining: Harvest cells carefully, wash in PBS with 2% FBS

  • For intracellular staining: Fix cells with Flow Cytometry Fixation Buffer and permeabilize with Flow Cytometry Permeabilization/Wash Buffer

• Antibody staining:

  • Block with 2% FBS in PBS for 30 minutes on ice

  • Incubate with primary anti-LPHN2 antibody at optimized concentration

  • Wash 3× with PBS/2% FBS

  • Incubate with fluorophore-conjugated secondary antibody (e.g., Allophycocyanin-conjugated Anti-Mouse IgG)

  • Wash 3× and resuspend in appropriate buffer for analysis

• Controls:

  • Include isotype control antibody (e.g., MAB10041) stained cells

  • Include unstained cells for autofluorescence control

  • When possible, include LPHN2-negative and LPHN2-positive cell populations

This approach has been successfully used to detect LPHN2 in transfected HEK293 cells .

How can I address non-specific binding issues with LPHN2 antibodies?

To reduce non-specific binding:

• Antibody dilution optimization: Titrate antibody concentration to find optimal signal-to-noise ratio

• Additional blocking: Include 0.1-0.3% Triton X-100 in blocking solution for better penetration

• Alternative blocking agents: Try different blockers (BSA, normal serum, commercial blockers) if milk proteins cause background

• Cross-adsorbed secondary antibodies: Use highly cross-adsorbed secondary antibodies to reduce species cross-reactivity

• Peptide competition: Pre-incubate primary antibody with immunizing peptide as a specificity control

Remember that even validated antibodies may show different performance across applications - an antibody performing well in Western blot may not be optimal for IHC without protocol optimization.

How can I use LPHN2 antibodies to study its role in mechanoelectrical transduction?

For MET function studies:

• Force application experiments:

  • Use antibody-coated paramagnetic beads (LPHN2-M-beads) that recognize the N-terminus of LPHN2

  • Apply magnetic force in a controlled manner to stimulate LPHN2

• MET response measurement:

  • Record MET currents in hair cells before and after LPHN2 antibody application

  • Test if specific inhibitors of LPHN2 block MET responses

• Co-localization studies:

  • Use LPHN2 antibodies together with markers for MET channel components

  • Examine if LPHN2 co-localizes with TMC1 and other MET proteins

• LPHN2 deficiency models:

  • Compare MET responses in wild-type versus LPHN2-deficient hair cells

  • Examine if LPHN2 antibody effects are absent in LPHN2-deficient cells

Research shows that LPHN2 is expressed at stereocilia tips where MET occurs, associates with MET channel components, and that LPHN2 deficiency causes hearing loss and impaired MET responses .

What approaches can I use to study LPHN2's role in neural circuit formation?

For neural circuit studies:

• Misexpression assays:

  • Use lentiviral vectors expressing Cre or Cre-LPHN2 injected into defined brain regions (e.g., CA1)

  • Follow with injection of Cre-dependent tracers (e.g., AAV-DIO-mCherry) to visualize axonal projections

• Mutant analysis:

  • Compare effects of wild-type LPHN2 with mutants lacking tethered agonist activity or autoproteolytic cleavage

  • Analyze axon targeting patterns to determine which LPHN2 functions are required in specific contexts

• Antibody localization:

  • Use anti-LPHN2 antibodies to confirm protein expression in axons via FLAG immunostaining

  • Compare endogenous versus ectopic expression levels

• Quantitative analysis:

  • Calculate fraction of axon intensity within defined regions (e.g., thirds of the subiculum)

  • Perform statistical analysis to assess significance of targeting differences

Research has shown that wild-type LPHN2 misexpression in proximal CA1 neurons causes their axons to mistakenly target the proximal subiculum rather than their normal target in the distal subiculum .

How can LPHN2 antibodies be used to study its potential therapeutic applications in diabetic conditions?

Recent research identifies LPHN2 as a receptor for LRG1 with therapeutic potential:

• Expression analysis:

  • Use anti-LPHN2 antibodies to measure LPHN2 expression in diabetic versus non-diabetic tissues

  • Pay particular attention to tissues like penile tissue that show significantly increased LPHN2 expression in diabetic models

• Signaling pathway exploration:

  • Use antibodies to detect activation of PI3K, AKT, and NF-κB p65 pathways downstream of LPHN2

  • Compare pathway activation before and after LRG1 administration

• Vascular and neurological assessment:

  • Use LPHN2 antibodies alongside vascular and neuronal markers to study co-localization in diabetic tissues

  • Examine changes in this pattern after therapeutic interventions

Research suggests that the LRG1/LPHN2 axis may provide mechanistic insights into angiogenesis and nerve regeneration in diabetes, with potential therapeutic applications for diabetic erectile dysfunction and other complications .

What technical considerations are important when using LPHN2 antibodies for co-immunoprecipitation studies?

For co-IP applications:

• Antibody selection:

  • Choose antibodies raised against epitopes unlikely to be involved in protein-protein interactions

  • Consider using tagged LPHN2 constructs and anti-tag antibodies as alternatives

• Crosslinking considerations:

  • For transient interactions, consider mild crosslinking before lysis

  • Use membrane-permeable crosslinkers like DSP for intact cell crosslinking

• Lysis conditions:

  • Use gentle detergents (0.5-1% NP-40 or Triton X-100) to maintain protein-protein interactions

  • Include protease inhibitors and phosphatase inhibitors if studying phosphorylation states

• Controls:

  • Include IgG control immunoprecipitations

  • Consider using LPHN2-deficient samples as negative controls

  • For interaction studies, include conditions that should disrupt the interaction

This approach could be used to study LPHN2's reported interactions with proteins like TMC1 and teneurin-3, which are important for its functions in MET and neural circuit formation .