LRIT1 Antibody

What is LRIT1 and why is it important in vision research?

LRIT1 is a leucine-rich repeat protein found at photoreceptor synapses that binds trans-synaptically to metabotropic glutamate receptor 6 (mGluR6), which is the principal receptor in postsynaptic ON-bipolar cells of the retina . Its importance lies in its role in modulating synaptic communication in the retina. Research has shown that LRIT1 knockout in mice increases the sensitivity of cone synaptic signaling while impairing adaptation to background light . This protein is particularly critical for visual function under conditions requiring temporally challenging discrimination of visual signals in steady background light, suggesting its importance in daylight vision adaptation mechanisms . Understanding LRIT1 is essential for researchers investigating retinal processing, synaptic plasticity, and visual adaptation mechanisms.

What applications are LRIT1 antibodies commonly used for?

Based on the available data, LRIT1 antibodies are primarily used in the following applications:

These applications enable researchers to investigate LRIT1 expression, localization, and interactions in various experimental models. For example, Western blotting has been used to confirm the specificity of LRIT1-mGluR6 interaction in native retinal tissues .

What epitopes of LRIT1 do commercially available antibodies typically target?

Commercial LRIT1 antibodies are primarily generated against specific amino acid sequences within the protein. Based on the search results, common epitope targets include:

• C-terminal region amino acids 594-622 of human LRIT1

• C-terminal region amino acids 601-623 of human LRIT1

• Middle region amino acids 251-456 of human LRIT1

These antibodies are typically generated from rabbits immunized with a KLH (Keyhole Limpet Hemocyanin) conjugated synthetic peptide corresponding to these regions . The choice of epitope can affect antibody specificity and performance in different applications, so researchers should select antibodies targeting epitopes suitable for their experimental needs.

What are the optimal conditions for using LRIT1 antibodies in Western blotting?

When using LRIT1 antibodies for Western blotting, researchers should consider the following methodological aspects:

• Sample preparation: LRIT1 is a transmembrane protein, so effective membrane protein extraction is crucial. Standard RIPA or NP-40 based lysis buffers with protease inhibitors are recommended.

• Antibody selection: Choose antibodies that have been validated for Western blot applications. For example, the antibody described in search result (ABIN1812566) and the antibody in search result (ABIN2303544) have both been validated for Western blotting.

• Dilution optimization: While specific optimal dilutions should be determined experimentally, a typical starting range for primary antibody incubation would be 1:500 to 1:2000 dilution.

• Positive control: Include retinal tissue lysate as a positive control, as LRIT1 is retina-specific .

• Detection system: An appropriate secondary antibody conjugated to HRP or fluorescent labels should be selected based on the host species (rabbit for most LRIT1 antibodies) .

• Expected band size: LRIT1 antibodies should detect a single band corresponding to the predicted size of the LRIT1 protein (approximately 69-71 kDa) .

For confirmation of specificity, knockout or knockdown controls can be used, as demonstrated in research where anti-LRIT1 antibodies detected a single band in wild-type but not in nob3 retinas (a model lacking LRIT1) .

How can I optimize immunohistochemical detection of LRIT1 in retinal tissues?

For optimal immunohistochemical detection of LRIT1 in retinal tissues, consider the following protocol optimizations:

• Fixation method: Use 4% paraformaldehyde for 15-30 minutes for optimal preservation of antigenic epitopes while maintaining tissue architecture.

• Antigen retrieval: This may be necessary to expose epitopes masked by fixation. Citrate buffer (pH 6.0) heat-induced epitope retrieval has been successful for many retinal proteins.

• Sectioning: Use either cryosections (10-12 μm) or vibratome sections (50-100 μm) depending on the desired analysis.

• Blocking: Block with 5-10% normal serum (from the species of secondary antibody) with 0.1-0.3% Triton X-100 for 1-2 hours to reduce non-specific binding.

• Primary antibody incubation: Incubate with LRIT1 antibody at optimized dilution (typically 1:100 to 1:500) overnight at 4°C.

• Counterstaining: Consider co-staining with synaptic markers (e.g., PSD95 for photoreceptor terminals) to confirm LRIT1 localization at photoreceptor synapses, as demonstrated in previous research .

• Negative controls: Include sections from LRIT1 knockout models or use secondary antibody only controls.

For confocal imaging, careful attention to z-stack acquisition parameters is important due to the precise synaptic localization of LRIT1 at photoreceptor terminals.

How can LRIT1 antibodies be used to investigate LRIT1-mGluR6 interactions in retinal circuits?

Investigating LRIT1-mGluR6 interactions using LRIT1 antibodies can be approached through several methodologies:

• Co-immunoprecipitation (Co-IP): This technique has been successfully used to demonstrate LRIT1-mGluR6 interactions in native retinas. Researchers can immunoprecipitate mGluR6 and then probe for LRIT1 using specific antibodies, or vice versa. Published work has confirmed the specificity of this interaction by showing a single band corresponding to LRIT1 in wild-type but not in nob3 retinas .

• Proximity Ligation Assay (PLA): This technique can be employed to visualize protein-protein interactions in situ with high specificity and sensitivity. Using antibodies against LRIT1 and mGluR6 from different host species, this approach could identify interaction sites within the retinal tissue.

• FRET-based assays: Using fluorescently labeled antibodies against LRIT1 and mGluR6, Förster Resonance Energy Transfer can be used to study these interactions in living cells or fixed tissues.

• Reconstituted systems: Following the approach described in the research, HEK293 cells can be co-transfected with LRIT1 and mGluR6 followed by immunoprecipitation experiments to verify direct interactions .

When designing these experiments, researchers should be careful about antibody compatibility and proper controls. For instance, using antibodies raised in different host species for co-IP or PLA is recommended to avoid cross-reactivity issues.

What are the key considerations when comparing LRIT1 and LRIT3 antibodies for studies of retinal ON bipolar cell signaling?

When comparing LRIT1 and LRIT3 antibodies for studies of retinal ON bipolar cell signaling, researchers should consider the following key factors:

• Distinct roles: While both proteins are members of the LRIT family, they perform different functions in retinal signaling. LRIT1 modulates adaptive changes in synaptic communication of cone photoreceptors , whereas LRIT3 is crucial for post-synaptic signaling complex (signalplex) assembly in ON bipolar cells .

• Expression patterns: LRIT1 is expressed in photoreceptors , while LRIT3 is expressed in both photoreceptors and ON bipolar cells . Antibodies should be validated for specificity to distinguish between these related proteins.

• Experimental models: For LRIT3 studies, rAAV-mediated expression models have been used to investigate rescue of cone DBC signalplex components . Similar approaches could be considered for LRIT1 functional studies.

• Functional readouts:

  • For LRIT1: Studies focus on cone synaptic signaling sensitivity and adaptation to background light

  • For LRIT3: Measurements focus on restoration of nyctalopin, TRPM1, mGluR6, GPR179, and RGS11 to the cone DBC signalplex

• Electrophysiological approaches: Different recording protocols may be optimal for each protein:

  • LRIT1 studies may focus on light adaptation protocols to assess cone function

  • LRIT3 studies often use ERG measurements to assess ON bipolar cell function

Understanding these distinctions is crucial when selecting and applying antibodies for comparative studies of these two proteins in retinal circuitry research.

How can LRIT1 antibodies be used to investigate the molecular mechanisms of daylight vision adaptation?

LRIT1 antibodies can be valuable tools for investigating molecular mechanisms of daylight vision adaptation through several experimental approaches:

• Immunohistochemical analysis under different light conditions: Researchers can use LRIT1 antibodies to examine potential changes in LRIT1 localization or expression levels under different light adaptation states. This involves exposing experimental animals to varying light intensities before tissue fixation and immunostaining.

• Biochemical analysis of LRIT1 post-translational modifications: Using LRIT1 antibodies in combination with phospho-specific antibodies or other post-translational modification detection methods can reveal how LRIT1 may be modified in response to changing light conditions.

• Investigating LRIT1-interactome changes: Combining LRIT1 antibodies with mass spectrometry approaches after immunoprecipitation can identify proteins that interact with LRIT1 in a light-dependent manner.

• Correlation with electrophysiological data: Immunohistochemical analysis using LRIT1 antibodies can be performed on tissues from animals that have undergone electrophysiological recording to correlate LRIT1 expression or localization with functional changes in visual adaptation.

Research has shown that LRIT1 is regulated by the neurotransmitter release apparatus that controls photoreceptor output, and knockout of LRIT1 impairs the ability of cones to adapt to background light . These findings suggest that LRIT1 antibodies can be particularly useful for investigating the molecular mechanisms underlying this adaptation process.

What are common issues when using LRIT1 antibodies and how can they be resolved?

Researchers may encounter several common issues when using LRIT1 antibodies. Here are potential problems and their solutions:

• Low or no signal in Western blot:

  • Possible cause: Insufficient protein extraction due to LRIT1 being a transmembrane protein.

  • Solution: Use stronger lysis buffers containing appropriate detergents (e.g., 1% SDS or combination of NP-40 and deoxycholate) with complete protease inhibitors.

  • Possible cause: Epitope masking during fixation or processing.

  • Solution: Try different fixation protocols or include a mild denaturation step before antibody incubation.

• Non-specific bands in Western blot:

  • Possible cause: Cross-reactivity with related proteins (LRIT family members).

  • Solution: Use antibodies targeting unique regions of LRIT1 (e.g., the C-terminal antibodies described in search results and ) and optimize antibody concentration.

  • Possible cause: Degradation products.

  • Solution: Ensure samples are kept cold, use fresh protease inhibitors, and minimize freeze-thaw cycles.

• High background in immunohistochemistry:

  • Possible cause: Non-specific binding of primary or secondary antibodies.

  • Solution: Increase blocking time (2+ hours), use higher concentrations of blocking agents, and optimize antibody dilutions.

  • Possible cause: Autofluorescence of retinal tissue.

  • Solution: Include an autofluorescence quenching step (e.g., Sudan Black B treatment) in the protocol.

• Inconsistent results between experiments:

  • Possible cause: Variability in expression levels due to light adaptation state.

  • Solution: Standardize light conditions before tissue collection and processing.

  • Possible cause: Antibody lot-to-lot variation.

  • Solution: Validate each new lot against a reference sample with known LRIT1 expression.

Including appropriate positive controls (wild-type retina) and negative controls (LRIT1 knockout tissue, if available) is essential for troubleshooting and validating results with LRIT1 antibodies.

How can specificity of LRIT1 antibodies be validated in research applications?

Validating the specificity of LRIT1 antibodies is crucial for ensuring reliable research results. Here are comprehensive methods for antibody validation:

• Genetic knockout/knockdown controls:

  • Use tissue from LRIT1 knockout mice as negative controls. Previous research demonstrated antibody specificity by showing a single band in wild-type but not in nob3 retinas (LRIT1-deficient) .

  • Alternative: Use siRNA or shRNA knockdown of LRIT1 in appropriate cell lines to create validated controls.

• Recombinant protein controls:

  • Test antibodies against purified recombinant LRIT1 protein in Western blots.

  • Perform blocking experiments where the antibody is pre-incubated with excess immunizing peptide before application to samples.

• Multiple antibody approach:

  • Use multiple antibodies targeting different epitopes of LRIT1 (e.g., antibodies targeting AA 594-622 and AA 601-623 ) and compare results.

  • Concordant results with different antibodies increase confidence in specificity.

• Cross-reactivity testing:

  • Test the antibody against related proteins, particularly other LRIT family members (LRIT2, LRIT3) to ensure specificity.

  • Consider heterologous expression systems where LRIT family members are individually expressed.

• Mass spectrometry validation:

  • Perform immunoprecipitation with the LRIT1 antibody followed by mass spectrometry analysis to confirm that the precipitated protein is indeed LRIT1.

• Application-specific validation:

  • For immunohistochemistry: Compare staining patterns with published LRIT1 mRNA expression patterns.

  • For Western blots: Verify that the observed band corresponds to the predicted molecular weight of LRIT1 (approximately 69-71 kDa).

These validation approaches should be documented and reported in research publications to enhance reproducibility and reliability of results obtained using LRIT1 antibodies.

How should researchers interpret differences in LRIT1 immunolabeling patterns between rod and cone synapses?

When interpreting differences in LRIT1 immunolabeling patterns between rod and cone synapses, researchers should consider several key factors:

• Functional differences in synaptic transmission: Rods and cones have distinct physiological roles in vision—rods function in dim light conditions while cones operate in bright light and provide color vision. LRIT1 has been shown to play a critical role in modulating adaptive changes in synaptic communication, particularly in cone photoreceptors . Therefore, differences in immunolabeling may reflect these functional specializations.

• Synaptic architecture interpretation: Rod and cone terminals (spherules and pedicles, respectively) have different morphologies and synaptic arrangements. When analyzing LRIT1 immunolabeling, researchers should:

  • Use co-labeling with specific markers for rod (e.g., rhodopsin) and cone (e.g., cone opsins) photoreceptors to confirm cell type

  • Employ synaptic markers (e.g., PSD95, ribeye) to precisely localize LRIT1 within the synaptic structure

  • Use high-resolution microscopy techniques (structured illumination or confocal microscopy) to resolve fine structural details

• Quantitative assessment approaches: To objectively compare immunolabeling between rod and cone synapses, researchers should:

  • Develop standardized image acquisition parameters

  • Establish defined regions of interest (ROIs) for analysis

  • Apply consistent thresholding criteria for signal detection

  • Use appropriate statistical methods to compare labeling intensity and distribution

• Physiological correlates: Interpret immunolabeling differences in the context of known physiological differences between rod and cone signaling, particularly regarding light adaptation mechanisms. Research has shown that LRIT1 knockout impairs the ability of cones to adapt to background light , suggesting its critical role in cone-specific visual processing.

Understanding these differences is particularly important when investigating vision in conditions that differentially affect rod versus cone function, such as during light adaptation or in retinal diseases with selective effects on different photoreceptor types.

What considerations are important when analyzing LRIT1 expression changes in disease models or experimental manipulations?

When analyzing LRIT1 expression changes in disease models or experimental manipulations, researchers should consider several important factors for accurate interpretation:

• Baseline expression normalization: Establish reliable baseline expression of LRIT1 in control samples using appropriate housekeeping genes or proteins for normalization in Western blots or qPCR. For immunohistochemistry, use consistent imaging parameters across all samples.

• Cell-type specific analysis: Since LRIT1 is primarily expressed in photoreceptors, changes in LRIT1 expression should be evaluated in the context of photoreceptor viability. Consider:

  • Co-labeling with photoreceptor markers to distinguish between true expression changes and photoreceptor loss

  • Using cell-type specific markers (e.g., cone opsins, rhodopsin) to determine if changes are specific to rods, cones, or both

• Temporal dynamics: Analyze LRIT1 expression at multiple time points during disease progression or after experimental manipulation to distinguish between:

  • Primary changes directly related to the manipulation/disease

  • Secondary changes occurring as adaptive or compensatory responses

  • Late-stage changes associated with photoreceptor degeneration

• Functional correlation: Correlate LRIT1 expression changes with functional assessments:

  • Electrophysiological measurements (ERG, especially photopic responses)

  • Visual behavioral tests that assess adaptation to changing light conditions

  • Assessments of synaptic transmission in the outer retina

• Molecular context: Examine LRIT1 expression changes in relation to its binding partners, particularly mGluR6 and other components of photoreceptor synaptic signaling machinery.

• Technical considerations: Be aware of potential confounding factors:

  • Fixation artifacts or processing differences between samples

  • Antibody lot variation over the course of long-term studies

  • Differences in tissue orientation or sectioning plane

Incorporating these considerations will help researchers distinguish pathologically relevant changes in LRIT1 expression from technical artifacts or secondary consequences of disease or experimental manipulation.

How can LRIT1 antibodies be used for developing therapeutic approaches for retinal diseases?

LRIT1 antibodies can contribute to therapeutic development for retinal diseases in several innovative ways:

• Target validation and disease mechanism elucidation:

  • LRIT1 antibodies can help establish LRIT1's role in specific disease processes by examining its expression and localization in patient samples or disease models.

  • Immunohistochemical analysis using these antibodies can reveal whether LRIT1 expression or distribution is altered in conditions affecting cone function or daylight vision.

• Biomarker development:

  • LRIT1 antibodies could be used to develop assays for measuring LRIT1 levels in accessible samples (e.g., aqueous humor) as potential biomarkers for disease progression or treatment response.

  • Changes in LRIT1 expression might be indicative of altered cone function before clinical symptoms appear.

• Therapeutic development guidance:

  • Mapping the precise binding interface between LRIT1 and mGluR6 using antibodies that recognize specific epitopes can inform the development of small molecules or peptides that could modulate this interaction.

  • Understanding how LRIT1 affects cone adaptation to background light could lead to treatments for conditions with impaired light adaptation.

• Assessment of gene therapy efficiency:

  • Similar to studies with LRIT3 , LRIT1 antibodies can be used to confirm successful protein expression following gene therapy approaches targeting LRIT1 in relevant animal models.

  • Immunohistochemistry using these antibodies can verify proper localization of the expressed protein to photoreceptor synapses.

• Monitoring disease progression and treatment response:

  • LRIT1 antibodies can be used to assess changes in protein expression or localization during disease progression or following therapeutic intervention.

  • This information can help establish optimal treatment windows and evaluate treatment efficacy at the molecular level.

Research indicates that LRIT1 plays a critical role in modulating synaptic communication and adaptation to light , suggesting that therapeutic approaches targeting this protein could benefit patients with conditions characterized by impaired visual adaptation and daylight vision deficits.

What are the emerging methods for multiplexed detection of LRIT1 and associated synaptic proteins?

Emerging methods for multiplexed detection of LRIT1 and associated synaptic proteins offer powerful approaches for comprehensive analysis of retinal synaptic complexes. Here are advanced techniques researchers should consider:

• Multiplex immunofluorescence techniques:

  • Sequential immunostaining: Using antibody stripping or quenching between rounds of staining to detect multiple proteins.

  • Spectral unmixing: Utilizing antibodies with overlapping fluorescence spectra, then computationally separating signals based on spectral signatures.

  • Tyramide signal amplification (TSA): Enabling detection of multiple antigens with antibodies from the same species by amplifying and permanently depositing fluorophores before antibody elution.

• Advanced imaging technologies:

  • Super-resolution microscopy: Techniques like STORM, PALM, or STED microscopy can resolve LRIT1 and binding partners (e.g., mGluR6 ) at the nanoscale level within the synaptic complex.

  • Expansion microscopy: Physical expansion of tissue samples can improve spatial resolution for mapping synaptic proteins.

  • Array tomography: Serial ultra-thin sections combined with multiplexed immunofluorescence for 3D reconstruction of synaptic structures.

• Proximity-based detection methods:

  • Proximity Ligation Assay (PLA): For visualizing protein-protein interactions between LRIT1 and its binding partners in situ.

  • FRET/FLIM analysis: For studying molecular interactions and conformational changes in living tissue.

  • HaloTag or SNAP-tag fusion proteins: Allowing specific labeling of proteins with synthetic fluorophores for live imaging.

• Mass spectrometry-based approaches:

  • Imaging mass cytometry: Combining immunohistochemistry with mass spectrometry for highly multiplexed analysis.

  • CODEX (CO-Detection by indEXing): Allowing detection of dozens of proteins in the same tissue section.

  • Proximity-dependent biotinylation (BioID/TurboID): For identifying proteins in close proximity to LRIT1 in the synaptic environment.

• Single-cell analysis methods:

  • Single-cell proteomics: Analyzing protein expression patterns in individual photoreceptors.

  • Spatial transcriptomics combined with protein detection: Correlating LRIT1 protein localization with gene expression patterns.

These emerging methods offer unprecedented opportunities to understand the molecular organization of photoreceptor synapses and how LRIT1 interacts with other components to regulate synaptic communication and adaptation mechanisms in vision .

What are the most promising future research directions for LRIT1 antibody applications in vision science?

Based on current knowledge about LRIT1 and available antibody technologies, several promising future research directions emerge:

• Systems neuroscience integration: LRIT1 antibodies could be used in large-scale mapping projects of the retinal connectome to understand how LRIT1-expressing synapses integrate into broader retinal circuits. This could reveal how LRIT1-mediated adaptive changes in cone synaptic signaling contribute to visual processing at the network level.

• Comparative species studies: Applying LRIT1 antibodies across different species with varying visual specializations (nocturnal vs. diurnal, color vision capabilities) could reveal evolutionary adaptations in LRIT1 expression and function. This comparative approach could uncover fundamental principles of visual adaptation mechanisms.

• Development and aging research: Tracking LRIT1 expression during retinal development and aging using stage-specific antibody labeling could provide insights into:

  • Critical periods for synapse formation and maturation

  • Changes in adaptational mechanisms throughout life

  • Potential therapeutic windows for intervention in developmental or age-related visual disorders

• Advanced humanized models: Validating LRIT1 antibodies for use in human retinal organoids or post-mortem tissues would enable translational research on:

  • Human-specific aspects of LRIT1 function

  • Pathological changes in retinal diseases

  • Preclinical testing of therapeutic approaches

• Synaptopathy mechanisms: Using LRIT1 antibodies to investigate retinal synaptopathies (synaptic disorders) could reveal whether alterations in LRIT1 expression or localization contribute to synaptic dysfunction in conditions like diabetic retinopathy or inherited retinal diseases.

• In vivo molecular imaging: Developing derivatives of LRIT1 antibodies (e.g., Fab fragments) conjugated to biocompatible fluorophores could potentially enable in vivo molecular imaging of LRIT1 dynamics in animal models, providing unprecedented insights into real-time changes in protein localization during light adaptation.

Research has established LRIT1's importance in modulating adaptive changes in photoreceptor synaptic communication , suggesting that these future directions could significantly advance our understanding of visual processing and adaptation mechanisms while potentially uncovering new therapeutic approaches for vision disorders.

What are the key unanswered questions regarding LRIT1 function that could be addressed using antibody-based approaches?

Several critical unanswered questions about LRIT1 function could be addressed using advanced antibody-based approaches:

• Regulation of LRIT1 expression and localization:

  • How does LRIT1 expression change under different light adaptation states?

  • Are there post-translational modifications of LRIT1 that regulate its function?

  • What trafficking mechanisms control LRIT1 localization to photoreceptor synapses?

  These questions could be addressed using phospho-specific antibodies, antibodies against modified forms of LRIT1, and high-resolution imaging with LRIT1 antibodies combined with markers of cellular trafficking machinery.

• Detailed structural interactions:

  • What is the precise binding interface between LRIT1 and mGluR6?

  • Are there other unidentified binding partners of LRIT1 at photoreceptor synapses?

  • How does the three-dimensional arrangement of LRIT1 and its partners change during light adaptation?

  Approaches using epitope-specific antibodies, proximity labeling with antibody-conjugated enzymes, and super-resolution microscopy could help resolve these structural questions.

• Cell-type specific functions:

  • Are there functional differences in LRIT1's role between different cone types (S, M, L cones)?

  • Does LRIT1 serve different functions in rods versus cones?

  • Are there species-specific differences in LRIT1 expression and function?

  Multiplexed immunohistochemistry with cone-specific markers and comparative studies across species using validated antibodies could address these questions.

• Disease-related alterations:

  • Is LRIT1 expression or localization altered in retinal diseases affecting daylight vision?

  • Could LRIT1 dysfunction contribute to specific visual symptoms in retinal disorders?

  • Are there genetic variants of LRIT1 that affect protein function or expression?

  Antibody-based screening of patient samples and disease models could help answer these clinically relevant questions.

• Developmental dynamics:

  • When during development is LRIT1 first expressed at photoreceptor synapses?

  • Does LRIT1 play a role in synapse formation or maturation?

  • How is LRIT1 expression regulated during retinal development?

  Developmental timecourse studies using LRIT1 antibodies could provide important insights into these aspects.