LRAT Antibody

What is LRAT and what is its biological significance?

LRAT (Lecithin retinol acyltransferase) is a membrane-bound enzyme that transfers the acyl group from the sn-1 position of phosphatidylcholine to all-trans retinol, producing all-trans retinyl esters which serve as storage forms of vitamin A . This enzyme plays a critical role in vision by providing the all-trans retinyl ester substrates for isomerohydrolase, which processes these esters into 11-cis-retinol in the retinal pigment epithelium . LRAT is required for the survival of cone photoreceptors and correct rod photoreceptor cell morphology . Beyond vision, LRAT is essential for dietary mobilization, transport, and storage of vitamin A in tissues such as the liver and lung . Additionally, LRAT can exchange palmitoyl groups between RPE65 (a tRE binding protein) and tREs, which is important for proper visual pathway functioning .

What types of LRAT antibodies are currently available for research?

Currently, researchers have access to several types of LRAT antibodies, with rabbit polyclonal antibodies being the most commonly documented in the literature . For example, Abcam offers a rabbit polyclonal LRAT antibody (ab137304) suitable for Western blotting applications that reacts with human samples . Similarly, Thermo Fisher Scientific provides a polyclonal LRAT antibody (PA5-36972) with >95% purity by SDS-PAGE . These antibodies typically recognize endogenous LRAT protein at molecular weights of approximately 25 kDa (monomer) and 50 kDa (dimer) . When selecting an antibody, researchers should verify the immunogen used; for instance, some are developed against synthetic peptides within human LRAT sequence .

What cellular localization pattern should I expect when using LRAT antibodies?

When using properly validated LRAT antibodies, you should expect to observe localization to the membrane of the endoplasmic reticulum (ER) . Studies examining LRAT membrane topology and subcellular localization have demonstrated that LRAT assumes a single membrane-spanning topology with an N-terminal cytoplasmic and C-terminal luminal orientation . This localization pattern was established through N-linked glycosylation scanning approach and protease protection assays . When performing immunofluorescence or immunohistochemistry experiments, co-localization with established ER markers would provide additional confirmation of proper antibody specificity and expected LRAT localization patterns.

How can I validate the specificity of an LRAT antibody?

Validating antibody specificity is crucial considering that approximately 50% of commercial antibodies fail to meet basic characterization standards . For LRAT antibodies, implement the following multi-step validation approach:

• Western blot verification: Confirm detection of bands at the expected molecular weights of 25 kDa (monomer) and 50 kDa (dimer)

• Negative controls: Use tissues or cell lines with known LRAT knockout or low expression

• Positive controls: Include tissues with high LRAT expression (retinal pigment epithelium, liver)

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to demonstrate signal reduction

• Orthogonal validation: Compare results with an alternative detection method (e.g., mRNA expression)

• Cross-reactivity assessment: Test in species other than the intended target to determine specificity

This comprehensive validation approach addresses the "antibody characterization crisis" that has led to questionable research results and significant financial losses in biomedical research .

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

When performing Western blotting with LRAT antibodies, researchers should follow these methodological guidelines for optimal results:

• Sample preparation: Extract proteins from tissues or cells using a membrane protein extraction buffer containing detergents suitable for hydrophobic proteins (e.g., RIPA buffer with 1% Triton X-100)

• Denaturation: Heat samples at 70°C rather than 95°C to prevent aggregation of membrane proteins

• Gel selection: Use 10-12% SDS-PAGE gels for optimal resolution of the 25 kDa monomer and 50 kDa dimer forms

• Transfer conditions: Employ wet transfer to PVDF membranes (preferred over nitrocellulose for hydrophobic proteins)

• Blocking: Use 5% BSA in TBST rather than milk (which can contain endogenous bioactive lipids that may interfere)

• Antibody dilution: Follow manufacturer recommendations (typically 1:1000 to 1:2000 for primary antibodies)

• Incubation: Overnight at 4°C for primary antibody binding to maximize specific signal

• Detection: Monitor both ~25 kDa and ~50 kDa bands, as LRAT forms dimers through disulfide bond formation

Control samples should include tissues known to express LRAT (retinal pigment epithelium, liver) and negative controls lacking LRAT expression.

How can I optimize immunohistochemistry protocols for LRAT detection in retinal tissues?

Detecting LRAT in retinal tissues requires special considerations due to the tissue's unique properties. Follow this optimized protocol:

• Fixation: Use 4% paraformaldehyde for 2-4 hours; avoid overfixation which can mask epitopes

• Tissue processing: Cryoprotect in 30% sucrose before embedding and sectioning to preserve retinal architecture

• Antigen retrieval: Perform heat-mediated antigen retrieval using citrate buffer (pH 6.0) to expose masked epitopes

• Permeabilization: Include 0.1-0.3% Triton X-100 in blocking buffer to facilitate antibody access to membrane-associated LRAT

• Blocking: Block with 10% normal serum from the species of the secondary antibody plus 1% BSA

• Primary antibody: Incubate with LRAT antibody at 1:100-1:500 dilution overnight at 4°C

• Controls: Include sections with primary antibody omitted and peptide-blocked antibody controls

• Counterstain: Use DAPI for nuclear visualization and specific retinal cell markers to contextualize LRAT localization

• Imaging: Employ confocal microscopy for precise subcellular localization assessment

This protocol accounts for LRAT's endoplasmic reticulum membrane localization and topology .

What controls are essential when using LRAT antibodies in experimental protocols?

Implementing rigorous controls is crucial when working with LRAT antibodies, especially given concerns about antibody characterization in biomedical research . Essential controls include:

• Positive tissue controls: Samples known to express LRAT (retinal pigment epithelium, liver)

• Negative tissue controls: Samples with minimal LRAT expression or LRAT knockout models

• Technical negative controls: Omission of primary antibody while maintaining all other protocol steps

• Peptide competition/blocking controls: Pre-incubation of antibody with immunizing peptide to confirm specificity

• Isotype controls: Using non-specific antibodies of the same isotype, host species, and concentration

• Orthogonal validation: Correlate protein detection with mRNA expression (RT-PCR, RNA-seq, or in situ hybridization)

• Multiple antibody validation: Use at least two antibodies recognizing different epitopes of LRAT

• Recombinant protein standards: Include purified LRAT protein (when available) as a size reference

These controls address the antibody reproducibility crisis that has been estimated to cause financial losses of $0.4-1.8 billion per year in the United States alone .

How should I approach co-immunoprecipitation experiments with LRAT antibodies?

Co-immunoprecipitation (Co-IP) of LRAT presents challenges due to its membrane localization. Here's a methodological approach:

• Lysis buffer selection: Use a buffer containing 1% digitonin or 1% DDM (n-dodecyl β-D-maltoside) to solubilize membrane proteins while preserving protein-protein interactions

• Pre-clearing: Pre-clear lysates with Protein A/G beads to reduce non-specific binding

• Antibody coupling: Consider covalently coupling LRAT antibody to beads using cross-linking reagents to prevent antibody contamination in eluates

• Immunoprecipitation conditions: Perform overnight incubation at 4°C with gentle rotation

• Washing stringency: Use incremental washing stringency to find optimal conditions that maintain specific interactions

• Elution method: Consider native elution using competing peptides rather than denaturing elution

• Detection strategy: Probe for known or suspected interaction partners such as RPE65, as LRAT can exchange palmitoyl groups with this protein

• Controls: Include IgG control, input sample, and flow-through fractions in Western blot analysis

Remember that LRAT monomers interact to form homodimers through disulfide bonding , so reducing conditions during analysis will affect oligomerization state detection.

What approaches should I use for LRAT antibody-based tissue immunostaining?

For optimal LRAT immunostaining in various tissues, follow these methodological guidelines:

• Tissue selection: Focus on tissues with known LRAT expression (retina, liver, lung) as positive controls

• Fixation optimization: Compare aldehyde-based fixatives (paraformaldehyde) with alcohol-based fixatives to determine optimal epitope preservation

• Sectioning technique: For retinal tissue, orient samples properly to visualize all retinal layers; 10-12 μm sections are typically optimal

• Antigen retrieval methods: Test both heat-induced (citrate buffer, pH 6.0) and enzymatic antigen retrieval

• Antibody dilution series: Perform a titration experiment (1:50 to 1:500) to determine optimal signal-to-noise ratio

• Incubation conditions: Extended incubation (overnight at 4°C) often yields better results than short incubations

• Detection systems: For low abundance, use tyramide signal amplification or high-sensitivity polymer detection systems

• Counterstaining strategy: Combine with markers for subcellular compartments to confirm ER localization

• Multi-channel imaging: Co-stain with cell-type specific markers to identify exact cellular distribution

Document the precise protocol conditions that yield optimal results to ensure reproducibility across experiments.

How can LRAT antibodies be used to study retinoid cycle defects in vision disorders?

LRAT antibodies serve as powerful tools for investigating retinoid cycle defects in vision disorders through several advanced approaches:

• Comparative expression analysis: Quantify LRAT protein levels in healthy versus diseased retinal tissues using Western blotting with well-validated antibodies

• Spatial distribution mapping: Perform high-resolution immunohistochemistry to detect altered LRAT localization patterns in retinal disease models

• Protein-protein interaction studies: Use co-immunoprecipitation with LRAT antibodies to identify changes in interaction partners in disease states

• Functional correlation: Combine LRAT protein detection with enzymatic activity assays to relate expression changes to functional outcomes

• Temporal expression patterns: Track LRAT expression during disease progression using longitudinal sampling

• Therapeutic response monitoring: Assess LRAT expression changes following experimental treatments

• Genetic modifier analysis: Compare LRAT protein levels across patients with the same primary mutation but different disease severity

This approach is particularly relevant given that loss of LRAT correlates with early-onset severe retinal dystrophy and severe retinyl ester deprivation . LRAT's role in providing all-trans retinyl ester substrates for the isomerohydrolase, which processes esters into 11-cis-retinol in the retinal pigment epithelium, makes it a critical target for vision research .

What experimental strategies can help determine LRAT membrane topology?

Building upon previous findings that LRAT is localized to the endoplasmic reticulum with an N-terminal cytoplasmic/C-terminal luminal orientation , researchers can employ these experimental strategies:

• N-linked glycosylation scanning: Introduce consensus glycosylation sites at various positions and assess glycosylation status to determine luminal exposure

• Protease protection assays: Treat intact microsomes with proteases to determine which regions are accessible

• Fluorescence protease protection (FPP): Express LRAT with fluorescent tags at different termini and monitor fluorescence after protease treatment

• Cysteine accessibility methods: Introduce cysteine residues at various positions and test their accessibility to membrane-impermeable sulfhydryl reagents

• Antibody epitope mapping: Use antibodies against different LRAT regions in intact versus permeabilized cells

• Fluorescence resonance energy transfer (FRET): Measure interactions between labeled domains to infer relative positions

• Cryo-electron microscopy: For higher-resolution structural analysis of membrane integration

These methods can refine our understanding of how LRAT's single membrane-spanning topology relates to its function in vitamin A metabolism and retinyl ester formation.

How can LRAT antibodies facilitate research on vitamin A metabolism beyond vision?

While LRAT's role in vision is well-established, its functions in other tissues offer important research opportunities:

• Tissue expression profiling: Use LRAT antibodies to create a comprehensive atlas of expression across multiple organs beyond the retina and liver

• Cancer research applications: Investigate the relationship between reduced LRAT expression and invasive bladder cancer using antibody-based detection methods

• Developmental studies: Track LRAT expression during embryonic development to understand vitamin A metabolism in organogenesis

• Nutritional intervention studies: Monitor LRAT protein levels in response to dietary vitamin A manipulation

• Stem cell differentiation: Evaluate LRAT as a potential marker for certain differentiation pathways dependent on retinoid signaling

• Lung development research: Examine LRAT's role in pulmonary surfactant metabolism and lung maturation

• Liver disease models: Assess LRAT expression changes in hepatic fibrosis or steatosis

• Immune cell function: Investigate potential roles in immune cells where retinoid signaling affects differentiation

These applications require antibodies with validated cross-reactivity in multiple tissue types and species to ensure reliable results across diverse experimental contexts.

What are the emerging technologies for developing more specific LRAT antibodies?

The field of antibody development is experiencing significant technological advancements that could improve LRAT antibody specificity:

• Deep learning-based design: Computational generation of antibody sequences with desirable developability attributes using training datasets of human antibodies

• In-silico antibody libraries: Creation of highly human antibody variable regions with intrinsic physicochemical properties resembling marketed antibody-based biotherapeutics

• Single B-cell cloning: Isolation of B cells producing antibodies against specific LRAT epitopes with subsequent sequencing and recombinant expression

• Phage display with synthetic libraries: Development of fully human antibodies with high specificity against LRAT epitopes

• CRISPR-engineered immunization models: Creation of animals expressing human LRAT sequences for more relevant immunization

• Structurally guided antibody engineering: Using LRAT structural information to design antibodies targeting specific functional domains

• Recombinant antibody fragments: Development of single-chain variable fragments (scFvs) with enhanced tissue penetration

• Multi-parameter screening platforms: High-throughput methods to simultaneously assess specificity, affinity, and developability

These approaches address the antibody characterization crisis by generating better characterized, more consistent reagents. The ability to computationally generate developable human antibody libraries represents a first step toward enabling in-silico discovery of antibody-based biotherapeutics .

What methodological approaches can detect post-translational modifications of LRAT?

Investigating post-translational modifications (PTMs) of LRAT requires specialized antibody-based approaches:

• Modification-specific antibodies: Develop antibodies specifically targeting phosphorylated, glycosylated, or palmitoylated forms of LRAT

• Two-dimensional gel electrophoresis: Separate LRAT protein forms based on both molecular weight and isoelectric point before antibody detection

• Phosphorylation site mapping: Use phospho-specific antibodies against predicted phosphorylation sites in LRAT

• Glycosylation analysis: Combine glycosidase treatments with Western blotting to identify glycosylated forms

• Chemical labeling strategies: Use bioorthogonal chemistry to tag specific modifications before antibody-based pulldown

• Mass spectrometry validation: Confirm antibody-detected modifications using mass spectrometry of immunoprecipitated LRAT

• In vitro enzymatic assays: Treat purified LRAT with kinases, phosphatases, or glycosylation enzymes before antibody detection

• Cellular signaling studies: Examine how different stimuli affect LRAT modification status

These approaches are particularly relevant given LRAT's role in exchanging palmitoyl groups and its membrane localization that may subject it to regulatory modifications.

Why might I observe unexpected band patterns when using LRAT antibodies in Western blotting?

Unexpected band patterns with LRAT antibodies may result from several factors requiring systematic troubleshooting:

Remember that LRAT monomers interact to form homodimers through disulfide bond formation , so reducing conditions will affect the detection of the 50 kDa dimer form.

How can I address low signal issues when using LRAT antibodies in immunofluorescence?

Low signal in immunofluorescence experiments with LRAT antibodies can be addressed through this methodological troubleshooting workflow:

• Fixation optimization: Overfixation can mask epitopes; try reduced fixation time or alternative fixatives

• Antigen retrieval enhancement: Test different antigen retrieval methods (heat-induced vs. enzymatic)

• Permeabilization improvement: Increase detergent concentration (0.1% to 0.3% Triton X-100) to better access the ER membrane where LRAT resides

• Antibody concentration adjustment: Prepare a dilution series to determine optimal concentration

• Incubation time extension: Increase primary antibody incubation to overnight at 4°C

• Detection system amplification: Implement tyramide signal amplification or high-sensitivity detection systems

• Blocking buffer modification: Test different blocking agents (normal serum, BSA, commercial blockers)

• Secondary antibody optimization: Use highly cross-adsorbed secondary antibodies to reduce background

• Microscopy settings enhancement: Optimize exposure, gain, and confocal laser settings

Document all optimization steps systematically to develop a reproducible protocol for future experiments.

What approaches help determine if my LRAT antibody is detecting the correct target?

Confirming target specificity is crucial given that approximately 50% of commercial antibodies fail to meet basic standards for characterization . Use these approaches:

• Genetic validation: Test antibody in LRAT knockout models or cells with CRISPR-mediated LRAT deletion

• siRNA knockdown: Compare antibody signal in cells treated with LRAT-targeting siRNA versus control siRNA

• Overexpression confirmation: Detect increased signal in cells overexpressing tagged LRAT construct

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry identification

• Epitope mapping: Determine the exact epitope recognized by the antibody through peptide arrays

• Cross-species reactivity: Test antibody against LRAT from multiple species with known sequence differences

• Peptide competition: Pre-absorb antibody with immunizing peptide to demonstrate signal elimination

• Signal correlation: Compare protein detection with mRNA expression across multiple tissue/cell types

This multi-method validation approach helps address the "antibody characterization crisis" that has led to questionable results in many scientific papers .

How do I interpret conflicting results between different LRAT antibodies?

Conflicting results between different LRAT antibodies require careful analysis and experimental design:

• Epitope mapping analysis: Determine if antibodies recognize different epitopes that might be differentially accessible

• Validation strength assessment: Evaluate the extent of validation for each antibody (publications, manufacturer data)

• Application-specific optimization: Some antibodies work better in certain applications (WB vs. IHC)

• Protocol compatibility evaluation: Test if antibodies require different sample preparation methods

• Cross-reactivity investigation: Assess potential cross-reactivity with related proteins

• Lot-to-lot variation consideration: Check if antibodies from different production lots were used

• Tissue-specific differences examination: Some epitopes may be masked in certain tissues due to protein interactions

• Isoform specificity determination: Confirm which LRAT isoforms or modified forms each antibody detects

Creating a comprehensive comparison table of antibody characteristics and performance across multiple experiments can help resolve these conflicts.

What strategies can distinguish between monomeric and dimeric forms of LRAT in experimental systems?

Distinguishing between the 25 kDa monomeric and 50 kDa dimeric forms of LRAT requires specific experimental strategies:

• Non-reducing vs. reducing conditions: Compare Western blots run under both conditions to preserve or disrupt disulfide bonds

• Sequential extraction methods: Use increasingly stringent detergents to differentially extract monomeric vs. dimeric forms

• Crosslinking experiments: Apply membrane-permeable crosslinkers before lysis to stabilize native dimeric associations

• Blue native PAGE: Perform native gel electrophoresis to maintain protein complexes before antibody detection

• Size exclusion chromatography: Fractionate lysates by size before immunoblotting to separate monomers and dimers

• Co-immunoprecipitation: Perform LRAT self-IP to confirm dimerization under different conditions

• FRET-based approaches: Use fluorescently tagged LRAT constructs to detect dimerization in live cells

• Chemical modification: Use sulfhydryl-reactive agents to block free cysteines before analysis

Understanding the monomer-dimer equilibrium may provide insights into LRAT function, as dimerization through disulfide bond formation likely affects enzymatic activity.

What are the most significant limitations of current LRAT antibody research?

Current LRAT antibody research faces several important limitations that researchers should consider:

• Epitope accessibility challenges: LRAT's membrane localization in the ER makes certain epitopes difficult to access with antibodies

• Limited isoform specificity: Most antibodies cannot distinguish between potential LRAT splice variants or modified forms

• Cross-species reactivity constraints: Many antibodies show limited validation across different model organisms

• Insufficient validation documentation: Many commercial antibodies lack comprehensive validation data specific to LRAT detection

• Monoclonal availability limitations: Most available LRAT antibodies are polyclonal , with fewer well-characterized monoclonal options

• Antibody reproducibility concerns: The broader "antibody characterization crisis" affects LRAT research reliability

• Functional correlation gaps: Limited tools to simultaneously assess LRAT protein presence and enzymatic activity

• Spatial resolution limitations: Difficulty in precisely localizing LRAT within membrane microdomains

These limitations reflect the broader challenges in antibody-based research, where an estimated 50% of commercial antibodies fail to meet basic standards for characterization .

How is computational antibody design revolutionizing LRAT research?

Deep learning approaches are transforming antibody development with potential applications for LRAT research:

• In-silico antibody generation: Computational models like Generative Adversarial Networks (GANs) can design antibody variable regions with desired properties

• Medicine-likeness optimization: Algorithms now generate antibodies whose physicochemical properties resemble marketed antibody therapeutics

• Developability prediction: Computational screening can identify sequences with favorable expression, stability, and solubility profiles

• Humanness assessment: Algorithms can ensure >90% humanness in generated antibody sequences, reducing immunogenicity concerns

• Diversity generation: Computational approaches can create diverse complementarity-determining regions (CDRs) capable of recognizing various antigens

• Experimental validation integration: In-silico predictions are now validated by measuring expression, monomer content, and thermal stability

• Reduced animal use: Computational design may eventually reduce reliance on animal immunization for antibody generation

These advances could lead to better-characterized LRAT antibodies and accelerate research by expanding the druggable antigen space to include targets refractory to conventional antibody discovery methods .

What emerging technologies will enhance LRAT functional studies?

Several emerging technologies promise to advance functional studies of LRAT:

• CRISPR-based genetic screens: Systematic modification of LRAT and related genes to understand functional networks

• Nanobody development: Generation of smaller antibody-derived molecules with enhanced accessibility to LRAT epitopes

• Proximity labeling techniques: BioID or APEX2 fusions to identify proximal proteins in the LRAT microenvironment

• Single-molecule imaging: Track individual LRAT molecules in membranes to understand dynamics and interactions

• Organoid models: Study LRAT function in physiologically relevant 3D retinal organoids

• Patient-derived iPSCs: Investigate LRAT dysfunction in cells from patients with retinal diseases

• Optogenetic regulation: Control LRAT activity with light to study temporal aspects of retinoid metabolism

• Cryo-electron tomography: Visualize LRAT in its native membrane environment at higher resolution

These approaches will help address mechanistic questions about how LRAT's membrane topology and dimerization affect its enzymatic function in various tissues.

How can researchers contribute to addressing the "antibody characterization crisis" in LRAT studies?

Researchers working with LRAT antibodies can help address the broader antibody characterization crisis through these practices:

• Comprehensive validation reporting: Document and publish all validation experiments performed with LRAT antibodies

• Resource sharing: Contribute validated antibodies and protocols to repositories and databases

• Knockout validation: Use CRISPR/Cas9 to generate LRAT knockout controls for definitive antibody validation

• Rigorous controls implementation: Include and report all positive and negative controls in publications

• Method transparency: Provide detailed methods sections with exact antibody catalog numbers, lots, and dilutions

• Recombinant standards use: Express and purify recombinant LRAT for antibody validation

• Alternative detection methods: Confirm key findings with orthogonal approaches not relying on antibodies

• Pre-registration of protocols: Consider pre-registering validation protocols to reduce selection bias

These practices address the problem that inadequately characterized antibodies cast doubt on reported results and contribute to financial losses estimated at $0.4–1.8 billion per year in the United States alone .

What future directions exist for combining LRAT antibodies with other research tools?

Integrating LRAT antibodies with complementary research tools offers promising future directions:

• Multi-omics integration: Combine antibody-based proteomics with transcriptomics and metabolomics for comprehensive analysis

• High-content screening approaches: Use automated microscopy with LRAT antibodies to screen compound libraries

• Tissue clearing techniques: Apply expanded sample transparency methods with LRAT immunostaining for 3D visualization

• Spatial transcriptomics correlation: Relate LRAT protein localization to spatial gene expression patterns

• LRAT-targeting antibody-drug conjugates: Develop therapeutic approaches for conditions with LRAT overexpression

• Microfluidic antibody analysis: Implement lab-on-chip approaches for rapid LRAT detection in small samples

• Machine learning image analysis: Apply AI algorithms to quantify subtle changes in LRAT distribution patterns

• Extracellular vesicle analysis: Investigate potential LRAT presence in exosomes as biomarkers

By combining antibody detection with these complementary approaches, researchers can gain deeper insights into LRAT's role in vitamin A metabolism and retinal physiology while addressing the limitations of any single methodology.