LHFPL5 Antibody, FITC conjugated

What is LHFPL5 and why is it important in auditory research?

LHFPL5 (Lipoma HMGIC fusion partner-like 5 protein) functions as an auxiliary subunit of the mechanotransducer (MET) non-specific cation channel complex located at the tips of the shorter stereocilia of cochlear hair cells. It plays a critical role in mechanotransduction by functionally coupling protocadherin 15 (PCDH15) to the transduction channel and mediating sensory transduction in the auditory system .

Research has demonstrated that LHFPL5 is essential for establishing maximal force transmission from the tip link to the MET channel. The MET complex consists of two dimeric pore-forming ion-conducting transmembrane TMC (TMC1 or TMC2) subunits, aided by several auxiliary proteins including LHFPL5, TMIE, CIB2/3, and TOMT, and the tip-link PCDH15 . Mutations in LHFPL5 are associated with non-syndromic sensorineural hearing loss in humans (DFNB67), mice, and zebrafish, highlighting its significance in hearing function .

What structural features of LHFPL5 are relevant for its function?

The LHFPL5 protein exhibits a tetraspan membrane structure with significant homology to claudins. Structurally, LHFPL5 contains:

• Four transmembrane domains (TM1-4)

• An N-terminal cytoplasmic domain

• Extracellular loops containing β-strands

• A β-sheet structure in the extracellular domain

• Two important disulfide bonds: one between Cys114 on TM2 and Cys130 on TM3, and another highly conserved bond between Cys68 on β3 and Cys79 on β4

What are the key technical specifications of LHFPL5 Antibody, FITC conjugated?

LHFPL5 Antibody, FITC conjugated is a high-quality polyclonal antibody with the following specifications:

These specifications are important for researchers to consider when designing experiments and interpreting results .

How can LHFPL5 Antibody, FITC conjugated be used for immunohistochemistry of cochlear tissues?

For immunohistochemistry of cochlear tissues using LHFPL5 Antibody, FITC conjugated, follow this optimized protocol:

• Tissue preparation:

  • Fix cochleae in 4% paraformaldehyde for 30 minutes at room temperature

  • After fixation, carefully dissect the organ of Corti to expose the hair cells

• Blocking and immunolabeling:

  • Incubate the fixed tissue in goat serum (as a blocking agent) for 1 hour at room temperature

  • Dilute the LHFPL5 Antibody, FITC conjugated at 1:100 to 1:200 in blocking buffer

  • Apply the antibody solution to the tissue and incubate overnight at 4°C in a humidified chamber

  • Co-stain with Alexa-568 phalloidin (1:500) for 1.5 hours to visualize actin-rich stereocilia

• Mounting and imaging:

  • Mount the immunolabeled cochleae with Fluoromount G

  • Image using a confocal microscope with a 60× objective (NA = 1.4) or 100× objective (NA = 1.45)

  • Measure fluorescence intensity using ImageJ (Fiji) software

This approach allows for visualization of LHFPL5 localization at the tips of stereocilia in hair cells, which is critical for understanding its role in mechanotransduction.

How can researchers distinguish between LHFPL5 and other members of the LHFP family in experiments?

Distinguishing LHFPL5 from other LHFP family members requires careful experimental design:

• Antibody selection:

  • Use antibodies specifically targeting unique epitopes of LHFPL5

  • For increased specificity, consider using antibodies targeting the N-terminal region of LHFPL5, which shows greater sequence divergence from other family members

• Controls for specificity validation:

  • Include Lhfpl5−/− knockout tissues as negative controls

  • Perform Western blot analysis to confirm antibody specificity

  • Consider transfection experiments with tagged versions of different LHFP family members (LHFPL2, LHFPL3, LHFPL4, and LHFPL5) to confirm antibody specificity

• Functional assays for distinction:

  • Utilize the unique functional properties of LHFPL5 in MET current rescue experiments

  • Research has shown that only LHFPL5 (not LHFPL2, LHFPL3, or LHFPL4) can rescue mechanotransduction currents in Lhfpl5−/− outer hair cells (OHCs), with currents at 1 μm deflection: 506.1 ± 41.8 pA for LHFPL5 versus 50.7-89.2 pA for other family members

• Protein interaction studies:

  • LHFPL5 uniquely binds to PCDH15, TMIE, and TMC1, which other LHFP family members do not

  • Immunoprecipitation experiments can be used to verify these specific protein-protein interactions

What methodological challenges exist when studying LHFPL5 localization in stereocilia?

Researchers face several methodological challenges when studying LHFPL5 localization in stereocilia:

• Fixation and preservation issues:

  • The delicate structure of stereocilia requires careful fixation to preserve morphology while maintaining antigen accessibility

  • Overfixation can reduce antibody penetration and epitope recognition

  • Recommendation: Use short fixation times (30 minutes) with 4% paraformaldehyde at room temperature

• Resolution limitations:

  • LHFPL5 localizes to the tips of the shorter stereocilia, requiring high-resolution imaging techniques

  • Conventional fluorescence microscopy may not provide sufficient resolution to precisely localize LHFPL5 within the MET complex

  • Solution: Use super-resolution microscopy techniques such as STORM, STED, or SIM for precise localization studies

• Quantification challenges:

  • Quantifying LHFPL5 expression levels at stereocilia tips requires standardized approaches

  • Recommendation: Use ratiometric analysis comparing LHFPL5 signal to a reference protein or structure, and employ consistent image acquisition parameters

• Sample orientation:

  • Proper orientation of cochlear samples is critical for accurate localization analysis

  • Solution: Use phalloidin counterstaining to visualize stereocilia and establish reference points for consistent orientation

• Age-dependent expression:

  • LHFPL5 expression and localization may vary during development

  • Methodological approach: Compare samples from defined developmental stages and document age-specific patterns

How can LHFPL5 antibodies be used to investigate the molecular architecture of the MET complex?

LHFPL5 antibodies, including FITC-conjugated versions, provide valuable tools for investigating the molecular architecture of the MET complex:

• Co-localization studies:

  • Use multi-color immunofluorescence with LHFPL5 antibodies and antibodies against other MET components (PCDH15, TMC1, TMIE)

  • This approach reveals spatial relationships between components and potential interaction domains

• Proximity ligation assays:

  • Employ proximity ligation assays (PLA) with LHFPL5 antibodies and antibodies against putative interaction partners

  • This method provides evidence of protein-protein interactions within 40 nm in native tissues

• Super-resolution microscopy:

  • Combine LHFPL5 antibodies with super-resolution techniques to map the precise arrangement of MET components

  • Research has shown that LHFPL5 forms a complex with PCDH15 where two LHFPL5 protomers interact via TM1 helices arranged in a V-shape, with PCDH15 transmembrane helices inserted into this V-shape

• Structural studies integration:

  • Correlate immunolocalization data with structural information from cryo-EM studies

  • The PCDH15-LHFPL5 complex has been visualized with a 2-fold symmetry, where LHFPL5 forms extensive interactions with PCDH15 transmembrane helices

• Functional correlation:

  • Combine localization studies with electrophysiological recordings to correlate protein arrangement with MET function

  • Research demonstrates that tip-link tension is transmitted to the channel primarily via LHFPL5, with residual activation without LHFPL5 occurring possibly by direct interaction

What are the most effective controls when using LHFPL5 Antibody, FITC conjugated in immunofluorescence studies?

For rigorous immunofluorescence studies using LHFPL5 Antibody, FITC conjugated, the following controls are essential:

• Genetic knockout controls:

  • Include tissues from Lhfpl5−/− knockout animals as negative controls

  • Lhfpl5+/− heterozygotes can serve as intermediate controls to assess dose-dependent effects

• Peptide competition controls:

  • Pre-incubate the antibody with excess immunogenic peptide (recombinant LHFPL5 protein) before application

  • This confirms binding specificity by demonstrating reduced or absent signal

• Alternative antibody validation:

  • Compare staining patterns with an independent LHFPL5 antibody targeting a different epitope

  • For example, compare results between antibodies targeting the N-terminal region and C-terminal region of LHFPL5

• Cross-reactivity assessment:

  • Include tissues known to express other LHFP family members but not LHFPL5

  • Non-specific binding would be indicated by signal in these tissues

• Multi-species validation:

  • When applicable, compare staining patterns across different species (mouse, zebrafish) known to express LHFPL5

  • Consistent localization patterns increase confidence in antibody specificity

• Signal-to-noise optimization:

  • Include controls with secondary antibody only (no primary antibody)

  • Test different antibody dilutions to optimize signal-to-noise ratio

  • For FITC-conjugated antibodies, include controls to assess autofluorescence in the same wavelength range

How can LHFPL5 Antibody be used to investigate mechanisms of deafness-causing mutations?

LHFPL5 Antibody can be strategically employed to investigate mechanisms of deafness-causing mutations through several approaches:

• Localization studies in mutant models:

  • Compare LHFPL5 localization in wild-type versus mutant tissues

  • Assess whether mutations affect trafficking of LHFPL5 to stereocilia tips

  • Research in mice shows that pathogenic mutations in LHFPL5 can disrupt proper localization of the protein, affecting MET function

• Protein interaction analyses:

  • Use co-immunoprecipitation with LHFPL5 Antibody to assess how mutations affect interactions with binding partners (PCDH15, TMC1, TMIE)

  • Chimeric studies between LHFPL5 and related proteins (e.g., LHFPL3) have revealed that the N-terminal half of LHFPL5 is required for binding to PCDH15, TMIE, and TMC1

• Functional correlation studies:

  • Combine immunolocalization with electrophysiological recordings to correlate protein distribution with functional defects

  • Studies have shown that in Lhfpl5−/− mice, the working range of transduction increases to 123 nm (from 52 nm in heterozygotes) and the single-channel gating force decreases to 0.13 pN (from 0.34 pN)

• Structure-function analysis:

  • Map deafness-causing mutations onto the known structural domains of LHFPL5

  • Specific mutations in the N-terminal cytoplasmic domain of LHFPL5 have been shown to disrupt optimal force sensitivity of the MET channel

• Rescue experiments:

  • Use LHFPL5 Antibody to verify expression of wild-type or mutant LHFPL5 in rescue experiments

  • This approach can confirm whether reintroduction of wild-type LHFPL5 restores proper localization and function

What methodological approaches are most effective for studying LHFPL5 interactions with other MET complex components?

To effectively study LHFPL5 interactions with other MET complex components, researchers should consider these methodological approaches:

• Co-immunoprecipitation studies:

  • Use LHFPL5 Antibody to pull down native protein complexes from hair cell lysates

  • Western blot analysis with antibodies against potential interaction partners (PCDH15, TMC1, TMIE) can confirm complex formation

  • When using heterologous expression systems, tag LHFPL5 with FLAG and co-express with potential binding partners for immunoprecipitation experiments

• FRET/FLIM analysis:

  • Utilize Förster Resonance Energy Transfer (FRET) between FITC-conjugated LHFPL5 Antibody and other fluorescently labeled components

  • This approach can detect protein-protein interactions with nanometer resolution in native tissues

• Chimeric protein analysis:

  • Generate chimeric proteins between LHFPL5 and related family members (e.g., LHFPL3)

  • Test these chimeras for interactions with MET components and functional rescue

  • Research has shown that chimeras with the N-terminal half of LHFPL5 (L5-L3) can bind efficiently to PCDH15 and TMIE, while both N- and C-terminal parts are required for efficient TMC1 binding

• Structural biology approaches:

  • Cryo-EM analysis of LHFPL5 with interaction partners can reveal molecular details of complex assembly

  • Studies have shown that the PCDH15-LHFPL5 complex consists of PCDH15 and LHFPL5 subunit pairs related by a 2-fold axis, with LHFPL5 forming extensive interactions with PCDH15 transmembrane helices

• Domain mapping:

  • Generate truncated or point-mutated versions of LHFPL5 to map interaction domains

  • For example, studies have shown that four amino acids in the N-terminal cytoplasmic domain of LHFPL5 are essential for establishing optimal force sensitivity of the MET channel through interactions with an amphipathic helix in TMC1

What are common challenges when using FITC-conjugated antibodies for cochlear immunohistochemistry, and how can they be addressed?

When using FITC-conjugated antibodies for cochlear immunohistochemistry, researchers often encounter these challenges with corresponding solutions:

• Tissue autofluorescence:

  • Challenge: Cochlear tissues often exhibit significant autofluorescence in the green spectrum, interfering with FITC signal

  • Solution: Treat samples with sodium borohydride (0.1% in PBS) for 30 minutes before blocking to reduce autofluorescence, or use Sudan Black B (0.1-0.3% in 70% ethanol) after immunolabeling

• Signal photobleaching:

  • Challenge: FITC is relatively prone to photobleaching during imaging

  • Solution: Use anti-fade mounting media containing DABCO or propyl gallate, minimize exposure during imaging, and consider sequential acquisition starting with the FITC channel

• Penetration issues:

  • Challenge: Limited antibody penetration into densely packed stereocilia bundles

  • Solution: Consider mild detergent treatment (0.1-0.3% Triton X-100) and/or perform extended incubation periods (48-72 hours) at 4°C

• pH sensitivity:

  • Challenge: FITC fluorescence is optimized at pH 8.0 and decreases at lower pH

  • Solution: Ensure all buffers are maintained at pH 7.8-8.2 for optimal FITC fluorescence

• Cross-reactivity:

  • Challenge: Polyclonal antibodies may exhibit cross-reactivity with related proteins

  • Solution: Include genetic controls (Lhfpl5−/− tissues) and perform blocking with non-immune serum from the same species as the secondary antibody

• Signal amplification:

  • Challenge: Weak FITC signal at endogenous expression levels

  • Solution: Consider tyramide signal amplification (TSA) to enhance detection sensitivity while maintaining specificity

How can researchers optimize LHFPL5 antibody-based protocols for different developmental stages of the auditory system?

Optimizing LHFPL5 antibody-based protocols for different developmental stages requires stage-specific adaptations:

• Embryonic and early postnatal stages:

  • Fixation: Use milder fixation (2% paraformaldehyde, 15-20 minutes) for improved antibody penetration

  • Tissue processing: Consider vibratome sectioning (50-100 μm) rather than whole-mount preparation

  • Antibody dilution: Use higher concentrations (1:50-1:100) due to lower expression levels

  • Incubation time: Extend primary antibody incubation to 48-72 hours at 4°C for better penetration

• Mid-postnatal stages (P5-P12):

  • This critical period for MET complex assembly requires balanced protocols:

  • Fixation: 4% paraformaldehyde for 20-30 minutes at room temperature

  • Tissue dissection: Careful removal of the tectorial membrane is essential without damaging stereocilia

  • Counterstaining: Include phalloidin to visualize developing stereocilia bundles

  • Analysis: Compare LHFPL5 distribution with developmental markers

• Mature auditory system:

  • Decalcification: For adult cochlea, add a gentle decalcification step (10% EDTA, pH 7.4, 1-3 days at 4°C)

  • Dissection: More vigorous mechanical dissection may be needed to expose hair cells

  • Antigen retrieval: Consider mild antigen retrieval (sodium citrate buffer, pH 6.0, 80°C for 30 minutes)

  • Controls: Age-matched controls are essential due to age-related changes in autofluorescence

• Age-specific quantification:

  • Normalize LHFPL5 expression to appropriate reference proteins at each developmental stage

  • Use consistent imaging parameters across age groups for valid comparisons

  • Document stereocilia maturation stage when reporting LHFPL5 localization patterns

• Species considerations:

  • For zebrafish studies, note that there are two LHFPL5 ohnologs (lhfpl5a and lhfpl5b)

  • Adjust fixation protocols for zebrafish tissues (3% paraformaldehyde, 10 minutes)

  • Validate antibody cross-reactivity with species-specific controls