LRIT1 Antibody, Biotin conjugated
Description
Introduction to LRIT1 Antibody, Biotin Conjugated
The LRIT1 Antibody, Biotin conjugated is a specialized immunological reagent designed to detect the Leucine-Rich Repeat, Immunoglobulin-Like, and Transmembrane Domains 1 (LRIT1) protein. LRIT1 is a transmembrane protein critical for synaptic communication in cone photoreceptors, particularly in light adaptation and visual signal processing . Biotin conjugation enhances the antibody's utility in assays requiring signal amplification, such as ELISA, Western blotting, and immunohistochemistry (IHC) .
Key Features of Biotin Conjugation
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Amplification: Biotin binds strongly to streptavidin or avidin, enabling signal enhancement in detection systems .
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Versatility: Compatible with enzymatic (e.g., HRP, AP) or fluorescent detection systems .
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Target specificity: Recognizes the extracellular leucine-rich repeat (LRR) domains of LRIT1 .
Applications of LRIT1 Antibody, Biotin Conjugated
The antibody is optimized for:
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ELISA: Quantitative detection of LRIT1 in lysates or tissue homogenates (sensitivity: 0.156 ng/ml) .
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Western Blotting: Immunodetection of LRIT1 in membrane fractions (size: ~60–70 kDa) .
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Immunohistochemistry: Localization of LRIT1 in photoreceptor synapses (outer plexiform layer) .
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Flow Cytometry: Surface staining of LRIT1 on transfected cells .
Role of LRIT1 in Visual Adaptation
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Light Adaptation Defects: LRIT1 knockout mice exhibit impaired cone-mediated visual responses under bright light conditions, as shown by reduced ERG b-wave amplitudes .
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Synaptic Modulation: LRIT1 interacts trans-synaptically with mGluR6, a key receptor in ON-bipolar cells, to regulate glutamate signaling .
Antibody Validation and Performance
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Specificity: The antibody (e.g., ABIN1910517) targets residues 594–622 of human LRIT1 and shows no cross-reactivity with other LRIT family proteins .
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Sensitivity: Detects LRIT1 in low-abundance samples via biotin amplification .
Experimental Use Cases
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
Validating LRIT1 Biotin-Conjugated Antibody Specificity Across Experimental Platforms
To confirm specificity:
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Perform knockout validation via Western blot using Lrit1−/− retinal lysates as negative controls .
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Use epitope mapping by comparing antibodies targeting different regions (e.g., AA 251-456 vs. AA 594-622) in parallel assays .
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Conduct blocking peptide competitions: Pre-incubate antibody with 10x molar excess of immunizing peptide (251-456AA recombinant protein or 594-622AA synthetic peptide) for 1 hr at 37°C before application .
Optimizing LRIT1 Detection in ELISA Assays
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Coating conditions: Use 2–4 μg/mL antibody in carbonate-bicarbonate buffer (pH 9.6) with overnight incubation at 4°C .
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Signal amplification: Pair biotinylated antibody with streptavidin-HRP (1:5,000 dilution) and TMB substrate.
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Cross-reactivity check: Include lysates from non-human primates or other species expressing LRIT1 homologs to assess interspecies reactivity .
Epitope Selection Criteria for Interaction Studies
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Domain-specific targeting:
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Structural considerations: Verify epitope accessibility via homology modeling using LRIT1’s predicted tertiary structure (UniProt Q6ZPR3).
Resolving LRIT1 Localization Discrepancies in Synaptic Studies
Problem: Conflicting reports of LRIT1 presynaptic (photoreceptor) vs. postsynaptic (bipolar cell) localization .
Solution:
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Perform dual-labeling super-resolution microscopy with anti-LRIT1 (AA 251-456) and synaptic markers (CtBP2 for ribbons, mGluR6 for ON-BCs) .
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Quantify colocalization using Manders’ coefficients (thresholded to >0.8 for true synaptic signal).
Controls for LRIT1-mGluR6 Interaction Assays
Essential controls:
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Genetic: Use mGluR6−/− (nob3) retinas in co-IP experiments .
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Pharmacological: Apply 100 μM LY341495 (mGluR6 antagonist) during co-IP to disrupt ligand-dependent interactions.
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Orthogonal validation: Reciprocal IP in HEK293T cells co-expressing LRIT1-FLAG and mGluR6-Myc .
Interpreting Phenotypes in LRIT1 Knockout Models
Contradiction: Increased cone BC sensitivity but impaired light adaptation .
Methodological framework:
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Electroretinography (ERG): Compare scotopic vs. photopic b-wave amplitudes under 0.1–10,000 cd/m² backgrounds .
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Single-cell electrophysiology: Patch-clamp cone BCs in retinal slices using 470 nm flashes (0.1–10,000 R*/cone/s).
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Quantitative Western blotting: Normalize LRIT1 levels to CaV1.4 (rod synapses) vs. ELFN1 (cone synapses) .
Table 1. Antibody Performance Across LRIT1 Domains
Table 2. LRIT1 Functional Parameters in Lrit1−/− Models
Technical Recommendations
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Multiplexing: Combine biotin-LRIT1 with FITC-conjugated CaV1.4 antibodies using sequential Avidin/Biotin blocking (Vector Labs).
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Fixation: For synaptic staining, use 4% PFA + 0.1% glutaraldehyde (10 min) to preserve LRIT1-mGluR6 complexes .
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Quantitative analysis: Apply Huygens Object Analyzer to distinguish true synaptic LRIT1 clusters from diffuse signal in IHC images.
