LRIT1 Antibody, HRP conjugated

What is LRIT1 and what is its biological significance?

LRIT1 (Leucine-rich repeat, immunoglobulin-like domain and transmembrane domain-containing protein 1) is a cell adhesion-like molecule that plays a critical role in visual processing. It is selectively enriched at cone photoreceptor synapses where it engages in a trans-synaptic interaction with mGluR6, the principal receptor in postsynaptic ON-bipolar cells . The protein features multiple extracellular modules including leucine-rich repeats (LRR), IgG-like domains, and fibronectin type III domains .

Research has demonstrated that LRIT1 is essential for modulating the sensitivity of cone synaptic signaling and its ability to adapt to background light. Knockout studies have shown that mice lacking LRIT1 display visual deficits under conditions requiring temporally challenging discrimination of visual signals in steady background light . This indicates LRIT1's fundamental role in scaling synaptic communication within the retina, particularly for daylight vision.

How is LRIT1 protein expression regulated in the retina?

LRIT1 expression and synaptic accumulation are inversely dependent on neurotransmitter release orchestrated by the CaV1.4 complex in photoreceptors. Studies have shown that deletion of pre-synaptic components (CaV1.4 and α2δ4) results in a dramatic elevation of LRIT1 expression, as demonstrated by both western blotting and immunohistochemical staining .

Interestingly, the regulation appears to be primarily from the presynaptic side, as deletion of postsynaptic components (mGluR6 or TRPM1) results in only minor downregulation of LRIT1 in the outer plexiform layer. This suggests a feedback mechanism where synaptic activity levels directly influence LRIT1 expression, positioning it as a potential mediator of adaptive responses in photoreceptor synapses .

What are the characteristics of commercially available LRIT1 Antibody, HRP conjugated?

The commercially available LRIT1 Antibody, HRP conjugated is a polyclonal antibody generated in rabbits using recombinant Human LRIT1 protein (amino acids 251-456) as the immunogen . Key characteristics include:

This antibody specifically recognizes human LRIT1 protein and comes directly conjugated to HRP, eliminating the need for secondary antibody incubation in certain applications .

What are optimal protocols for using LRIT1 Antibody, HRP conjugated in ELISA?

For optimal ELISA results with LRIT1 Antibody, HRP conjugated, researchers should consider the following protocol:

• Coating Phase: Coat ELISA plates with target antigen (recombinant LRIT1 or retinal tissue extract) at 1-10 μg/ml in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C.

• Blocking Phase: Block non-specific binding sites with 5% non-fat milk or 3% BSA in PBS-T (PBS + 0.05% Tween-20) for 1-2 hours at room temperature.

• Primary Antibody Incubation: Dilute LRIT1 Antibody, HRP conjugated at 1:1000 to 1:5000 in blocking buffer and incubate for 1-2 hours at room temperature or overnight at 4°C.

• Washing Steps: Wash the wells 4-5 times with PBS-T to remove unbound antibody.

• Detection: Add TMB substrate solution and monitor color development. Since the antibody is HRP-conjugated, no secondary antibody is needed .

• Signal Stopping: Stop the reaction with 2N H₂SO₄ when appropriate color develops.

• Measurement: Read absorbance at 450nm with a reference wavelength of 620nm.

For quantitative analysis, always include a standard curve using purified LRIT1 protein at known concentrations to calibrate your results.

How can I optimize Western blotting protocols for LRIT1 detection?

While the product information specifically mentions ELISA applications , HRP-conjugated antibodies can be adapted for Western blotting. Based on research practices with similar antibodies, consider the following optimization steps:

• Sample Preparation: Extract proteins from retinal tissue using RIPA buffer containing protease inhibitors. For membrane proteins like LRIT1, include 1% Triton X-100 to enhance solubilization.

• Gel Electrophoresis: Separate proteins on 10-12% SDS-PAGE gels.

• Transfer: Transfer proteins to PVDF or nitrocellulose membranes at 100V for 1 hour or 30V overnight.

• Blocking: Block membranes with 5% non-fat milk in TBS-T (TBS + 0.1% Tween-20) for 1 hour at room temperature.

• Primary Antibody: Dilute LRIT1 Antibody, HRP conjugated at 1:1000 to 1:5000 in blocking buffer and incubate overnight at 4°C.

• Washing: Wash membranes 3-4 times with TBS-T.

• Detection: Apply enhanced chemiluminescence (ECL) substrate directly to the membrane and detect signal using film or digital imaging systems .

• Expected Band Size: LRIT1 should appear at its predicted molecular weight (approximately 63 kDa for full-length human LRIT1).

To validate specificity, consider including positive controls (retinal tissue extracts) and negative controls (tissues known not to express LRIT1 or knockout models if available) .

What methodologies can be used to investigate LRIT1-mGluR6 interactions?

Based on published research on LRIT1-mGluR6 interactions, the following methodologies have proven effective:

• Co-immunoprecipitation (Co-IP): Immunoprecipitate mGluR6 from retinal lysates using anti-mGluR6 antibodies, then probe for LRIT1 in the eluates using anti-LRIT1 antibodies (or vice versa) . This approach has successfully demonstrated the specific interaction between these proteins in native retinas.

• Reconstituted System in HEK293 Cells: Co-transfect HEK293 cells with plasmids expressing mGluR6 and LRIT1, followed by reciprocal immunoprecipitation experiments. This system has shown robust and specific pull-down of LRIT1 when mGluR6 was immunoprecipitated and reciprocally mGluR6 upon LRIT1 immunoprecipitation .

• Proximity Ligation Assay (PLA): This technique can visualize protein-protein interactions in situ with high sensitivity. It would involve using antibodies against LRIT1 and mGluR6, followed by oligonucleotide-conjugated secondary antibodies that generate a fluorescent signal when the target proteins are in close proximity.

• FRET Analysis: Fluorescence Resonance Energy Transfer can be used to study the dynamic interaction between fluorescently tagged LRIT1 and mGluR6 in living cells.

• Immunohistochemical Co-localization: Using fluorescence microscopy with antibodies against LRIT1 and mGluR6 has shown partial overlap of these proteins in the synaptic cleft, consistent with their trans-synaptic interaction .

Each methodology provides complementary information about the nature, specificity, and dynamics of LRIT1-mGluR6 interactions.

What storage and handling practices ensure optimal antibody performance?

To maintain optimal activity of LRIT1 Antibody, HRP conjugated:

• Storage Temperature: Upon receipt, store the antibody at -20°C or -80°C . Avoid repeated freeze-thaw cycles by aliquoting the antibody into single-use volumes before freezing.

• Working Dilutions: Prepare working dilutions freshly on the day of the experiment whenever possible. If needed, store diluted antibody at 4°C for no more than 24 hours.

• Handling: Keep the antibody on ice during experiments. Avoid exposing HRP-conjugated antibodies to strong light as it may affect enzyme activity.

• Buffer Compatibility: The antibody is provided in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as preservative . When diluting, use buffers compatible with this formulation.

• Contamination Prevention: Always use clean pipette tips and tubes when handling the antibody to prevent contamination.

• Transportation: If transportation is necessary, ensure the antibody remains frozen using dry ice or similar cooling methods.

Adherence to these practices will help maintain antibody specificity and activity, ensuring consistent and reliable experimental results.

How can I troubleshoot high background or weak signals when using LRIT1 Antibody?

When encountering issues with LRIT1 Antibody, HRP conjugated, consider the following troubleshooting approaches:

For High Background:

• Increase Blocking Time/Concentration: Extend blocking time to 2 hours or increase BSA concentration to 5%.

• Optimize Antibody Dilution: Test a range of antibody dilutions (1:1000 to 1:10,000) to find the optimal signal-to-noise ratio.

• Add Detergent: Increase Tween-20 concentration in washing buffer to 0.1-0.3% to reduce non-specific binding.

• Additional Washing Steps: Implement more stringent washing protocols with increased number of washes and duration.

• Remove Contaminating Proteins: Pre-clear samples using Protein G agarose before immunoprecipitation or analysis.

For Weak Signals:

• Increase Protein Loading: For Western blots, increase the amount of total protein loaded per lane.

• Enhance Antigen Retrieval: For immunohistochemistry, optimize antigen retrieval methods (heat-induced or enzymatic).

• Extend Incubation Time: Increase primary antibody incubation time to overnight at 4°C.

• Check HRP Activity: Ensure the HRP conjugate is still active by testing with standard substrates.

• Enhance Detection Sensitivity: Use enhanced chemiluminescence (ECL) substrates with higher sensitivity for Western blotting.

• Fresh Sample Preparation: Ensure samples are freshly prepared with adequate protease inhibitors to prevent degradation of LRIT1.

Each experiment may require specific optimization based on the sample type, protein abundance, and detection method employed.

What controls should be included when working with LRIT1 Antibody?

Proper experimental controls are essential for interpreting results obtained with LRIT1 Antibody, HRP conjugated:

Positive Controls:

• Known LRIT1-expressing Tissue: Retinal tissue, particularly from the outer plexiform layer (OPL), which has been shown to have high LRIT1 expression .

• Recombinant LRIT1 Protein: Purified recombinant human LRIT1 protein can serve as a positive control for antibody specificity.

Negative Controls:

• LRIT1 Knockout Tissues: If available, tissues from LRIT1 knockout models (Lrit1−/−) can definitively demonstrate antibody specificity .

• Non-expressing Tissues: Tissues known not to express LRIT1 can serve as negative controls.

• Primary Antibody Omission: Omitting the primary antibody while keeping all other steps identical helps identify non-specific binding of detection reagents.

Technical Controls:

• Loading Controls: For Western blotting, include housekeeping proteins (β-actin, GAPDH) to normalize protein loading.

• Isotype Controls: Include irrelevant antibodies of the same isotype to assess non-specific binding.

• Dilution Series: Test multiple antibody dilutions to determine optimal concentration for the specific application.

Inclusion of these controls helps validate experimental results and troubleshoot any issues that may arise during the use of LRIT1 Antibody, HRP conjugated.

How can LRIT1 Antibody be used to study synaptic adaptations in photoreceptors?

LRIT1 Antibody can be utilized in several sophisticated approaches to investigate synaptic adaptations in photoreceptors:

• Quantitative Immunohistochemistry: Use the antibody to quantify changes in LRIT1 expression and localization under different light adaptation conditions. This approach can reveal how LRIT1 content at synapses changes in response to varying light stimuli, correlating molecular changes with functional adaptations .

• Comparative Analysis in Disease Models: Compare LRIT1 expression and localization between wild-type and retinal disease models to understand how synaptic adaptations are affected in pathological conditions.

• Activity-Dependent Regulation Studies: Investigate how manipulation of synaptic activity (through pharmacological agents or light stimulation protocols) affects LRIT1 levels. Research has shown that LRIT1 expression is inversely dependent on neurotransmitter release orchestrated by the CaV1.4 complex , suggesting it responds to activity-dependent signals.

• Time-Course Analysis During Development: Examine LRIT1 expression during different developmental stages to understand its role in the maturation of photoreceptor synapses.

• Co-localization with Synaptic Markers: Perform dual immunostaining with LRIT1 Antibody and antibodies against synaptic proteins (e.g., ribeye/CtBP2 for ribbons, mGluR6 for postsynaptic sites) to analyze the spatial relationship between LRIT1 and other synaptic components during adaptation .

These approaches can provide insights into how LRIT1 contributes to the dynamic scaling of synaptic transmission in response to changing light conditions.

What experimental design would best investigate the role of LRIT1 in cone-specific light adaptation?

Based on published research findings, an optimal experimental design to investigate LRIT1's role in cone-specific light adaptation would include:

• Comparative Electrophysiology: Record electroretinograms (ERGs) from wild-type and LRIT1-deficient retinas under various background light intensities. This approach has revealed that LRIT1 knockout impairs light adaptation specifically for cone-driven signals .

• Single-Cell Recordings: Perform patch-clamp recordings from cone ON-bipolar cells and cone OFF-bipolar cells in wild-type and LRIT1-deficient retinas to measure their sensitivity to light stimuli. Research has shown that cone bipolar cells in LRIT1 knockout mice are approximately 10-fold more sensitive than their wild-type counterparts .

• Visual Behavior Testing: Design behavioral tests that specifically assess cone-mediated visual functions under different adaptation states, comparing performance between wild-type and LRIT1-deficient animals.

• Molecular Dynamics Analysis: Use LRIT1 Antibody to examine changes in LRIT1 protein levels and localization during dark and light adaptation, correlating these changes with functional adaptations measured electrophysiologically.

• Calcium Imaging: Perform calcium imaging in photoreceptor terminals to visualize how calcium dynamics change during light adaptation in the presence or absence of LRIT1.

An experimental timeline might include:

• Baseline measurements in dark-adapted conditions

• Light adaptation at various intensities (targeting cone activation)

• Recovery period in darkness

• Analyses at molecular, cellular, and behavioral levels

This comprehensive approach would provide insights into how LRIT1 contributes to the adaptation mechanisms that allow cone photoreceptors to maintain responsiveness across a wide range of luminance changes.

How can researchers distinguish between LRIT1 effects in rod versus cone photoreceptor pathways?

Distinguishing LRIT1 effects between rod and cone pathways requires targeted experimental approaches:

• Selective Light Stimulation Protocols: Use stimuli that selectively activate either rod or cone photoreceptors. Research has shown that under scotopic light conditions (rod-dominated responses), LRIT1 knockout has minimal effects, while under photopic conditions (cone-dominated responses), significant differences are observed .

• Immunohistochemical Quantification: Use LRIT1 Antibody alongside markers that distinguish rod versus cone synapses to quantify LRIT1 enrichment. Studies have demonstrated that LRIT1 is more enriched in cone active zones compared to rods, suggesting a more prominent role in cone synaptic function .

• Electroretinography with Spectral Manipulation: Use colored light stimuli to preferentially activate different photoreceptor types (blue for S-cones, green for M-cones, etc.) while recording ERGs to dissect pathway-specific effects.

• Transgenic Approaches: Utilize mouse models with specific deficits in either rod or cone function to isolate LRIT1's role in each pathway.

• Ex Vivo Retinal Preparations: Perform pharmacological manipulations that selectively block either rod or cone pathways while assessing LRIT1-dependent functions.

• Visual Behavior Testing Under Different Light Conditions: Conduct behavioral tests under scotopic (rod-dominated) versus photopic (cone-dominated) lighting conditions to assess LRIT1's contribution to visual function in each photoreceptor pathway.

Through these methodological approaches, researchers can differentiate LRIT1's specific contributions to rod versus cone synaptic communication and adaptation mechanisms.

What are promising areas for future research on LRIT1 function in visual processing?

Based on current knowledge, several promising research directions could advance understanding of LRIT1's role in visual processing:

• Structural Biology Approaches: Determining the crystal structure of LRIT1 and its complex with mGluR6 would provide insights into the molecular basis of their interaction and potentially identify targets for therapeutic intervention.

• Human Visual Disorders: Investigating potential links between LRIT1 mutations or expression changes and specific visual disorders, particularly those involving deficits in light adaptation or daytime vision.

• Regulatory Mechanisms: Exploring the molecular mechanisms that regulate LRIT1 expression in response to changes in synaptic activity, including transcriptional, translational, and post-translational regulation.

• Development and Plasticity: Examining LRIT1's role in developmental processes and experience-dependent plasticity of retinal circuits.

• Comparative Studies Across Species: Investigating LRIT1 function across different species with varying visual capabilities could reveal evolutionary adaptations in synaptic communication mechanisms.

• Therapeutic Potential: Exploring whether modulation of LRIT1 function could provide therapeutic benefits for certain visual disorders, particularly those involving deficits in light adaptation.

• Interactions with Other Synaptic Proteins: Identifying additional binding partners of LRIT1 beyond mGluR6 could reveal broader roles in synaptic organization and function.

These research directions could significantly advance our understanding of how LRIT1 contributes to visual processing and potentially lead to new therapeutic approaches for visual disorders.

What technological advances might enhance LRIT1 research methodologies?

Emerging technologies that could significantly advance LRIT1 research include:

• Super-Resolution Microscopy: Techniques like STORM, PALM, or STED microscopy could provide unprecedented spatial resolution of LRIT1 localization at the synapse, revealing its precise distribution relative to other synaptic components.

• CRISPR/Cas9 Gene Editing: Generation of precise mutations or tagged versions of LRIT1 in relevant model systems could facilitate functional studies and protein tracking.

• Optogenetics and Chemogenetics: These approaches allow temporal control of neuronal activity, enabling precise manipulation of synaptic communication to study LRIT1's activity-dependent regulation.

• Single-Cell Transcriptomics and Proteomics: These techniques could reveal cell-type-specific expression patterns and regulatory networks involving LRIT1.

• In Vivo Imaging: Two-photon microscopy of fluorescently tagged LRIT1 in living retinal tissue could reveal dynamic changes in its distribution during adaptation processes.

• Cryo-Electron Microscopy: This technology could elucidate the molecular structure of LRIT1 and its complexes at near-atomic resolution.

• Computational Modeling: Integration of molecular, cellular, and systems-level data into computational models could predict how LRIT1 contributes to retinal circuit function under various conditions.

Implementation of these technologies would provide deeper insights into LRIT1's structural features, molecular interactions, and functional roles in visual processing.