LRAT Antibody, Biotin conjugated

What is LRAT and what biological functions does it serve?

LRAT (Lecithin retinol acyltransferase) is an enzyme that transfers the acyl group from the sn-1 position of phosphatidylcholine to all-trans retinol, producing all-trans retinyl esters which function as storage forms of vitamin A. LRAT plays a critical role in vision by providing the all-trans retinyl ester substrates for the isomerohydrolase that processes these esters into 11-cis-retinol in the retinal pigment epithelium. Through a membrane-associated alcohol dehydrogenase, 11-cis-retinol is oxidized and converted into 11-cis-retinaldehyde, which serves as the chromophore for rhodopsin and cone photopigments . LRAT defects are linked to severe early-onset retinal dystrophy, underscoring its importance in visual function .

What are the characteristics of biotin-conjugated LRAT antibody?

The biotin-conjugated LRAT antibody is a polyclonal IgG antibody typically raised in rabbit hosts against recombinant Human Lecithin retinol acyltransferase protein (amino acids 24-182) . The antibody is purified using Protein G affinity purification with purity >95%. The biotin conjugation allows for signal amplification in detection systems, as biotin can be recognized by avidin or streptavidin molecules, each capable of binding four biotin groups, thus enhancing detection sensitivity . The antibody is typically supplied in a buffer containing preservatives (0.03% Proclin 300), 50% glycerol, and 0.01M PBS at pH 7.4 .

What species reactivity does the LRAT antibody demonstrate?

While the specifications may vary between manufacturers, LRAT antibodies typically demonstrate reactivity with human samples . Some LRAT antibodies (non-biotin conjugated) have been validated to react with human, mouse, and rat samples . When selecting an LRAT antibody for your research, it is crucial to verify the specific species reactivity of the product you intend to use, especially if working with non-human models.

What are the validated applications for biotin-conjugated LRAT antibody?

The biotin-conjugated LRAT antibody has been validated for ELISA applications . The biotin conjugation makes this antibody particularly useful in avidin-biotin or streptavidin-biotin detection systems, where signal amplification can significantly enhance sensitivity. While the biotin-conjugated version is primarily validated for ELISA, unconjugated LRAT antibodies have been used successfully in Western blot applications , suggesting potential cross-application utility with appropriate protocol modifications.

How can biotin-conjugated LRAT antibody be used in immunohistochemistry studies?

Although the biotin-conjugated LRAT antibody in the search results is primarily validated for ELISA , biotin-conjugated antibodies generally can be utilized in immunohistochemistry through avidin-biotin or streptavidin-biotin detection systems. When adapting the LRAT antibody for IHC, researchers should consider:

• Antigen retrieval methods: Optimize based on tissue fixation (FFPE vs. frozen sections)

• Blocking endogenous biotin: Pre-treatment with avidin-biotin blocking reagents to prevent non-specific binding

• Detection system: Selection between ABC (Avidin-Biotin Complex) or streptavidin systems

• Visualization method: DAB, AP, or fluorescent-labeled streptavidin

A recommended starting dilution would be 1:200, with optimization required for specific tissue types and fixation methods .

How can biotin-conjugated LRAT antibody enhance detection sensitivity in ELISA assays?

Biotin-conjugated LRAT antibody enhances detection sensitivity in ELISA assays through the avidin/streptavidin-biotin amplification system. Both avidin and streptavidin are tetrameric proteins capable of binding 4 biotin groups per molecule, creating a significant amplification effect . This amplification mechanism increases the concentration of reporters at the antigenic site, substantially enhancing signal intensity.

For optimized ELISA protocols using biotin-conjugated LRAT antibody:

• Coat wells with capture antibody against LRAT

• Add sample containing LRAT protein

• Add biotin-conjugated LRAT antibody (1:500-1:1000 dilution)

• Add streptavidin-HRP or streptavidin-AP conjugate

• Add appropriate substrate for signal development

• Measure signal intensity using spectrophotometry

This method can achieve significantly higher sensitivity compared to direct detection methods, particularly advantageous when detecting low-abundance LRAT protein in biological samples.

What are the optimal storage conditions for maintaining biotin-conjugated LRAT antibody activity?

For optimal preservation of biotin-conjugated LRAT antibody activity, store the antibody at -20°C or -80°C upon receipt . Avoid repeated freeze-thaw cycles, as these can lead to protein denaturation and loss of binding activity. For regular use, consider aliquoting the antibody into smaller volumes before freezing to minimize freeze-thaw cycles. The presence of 50% glycerol in the storage buffer helps prevent freeze-thaw damage . For short-term storage (less than one month), the antibody can be kept at 2-8°C . Always centrifuge the antibody vial briefly before opening to ensure all liquid is at the bottom of the tube.

How should researchers optimize the dilution of biotin-conjugated LRAT antibody for different applications?

Dilution optimization is crucial for achieving optimal signal-to-noise ratio with biotin-conjugated LRAT antibody. For ELISA applications, typically start with a dilution range of 1:500-1:1000 . For other applications like immunohistochemistry or Western blotting (if attempting to adapt the biotin-conjugated antibody), begin with a dilution of 1:200 and adjust based on signal intensity and background levels .

To determine optimal dilution:

• Prepare a dilution series (e.g., 1:100, 1:200, 1:500, 1:1000, 1:2000)

• Test each dilution with positive and negative controls

• Evaluate based on:

  • Signal strength with positive control

  • Signal-to-noise ratio

  • Background staining with negative control

• Select the dilution that provides strong specific signal with minimal background

Remember that optimal dilution may vary between sample types and detection methods.

What controls should be included when working with biotin-conjugated LRAT antibody?

To ensure experimental validity when working with biotin-conjugated LRAT antibody, incorporate the following controls:

• Positive control: Samples known to express LRAT (e.g., retinal pigment epithelium, testis tissue , or Y79 retinoblastoma cells )

• Negative control: Samples known not to express LRAT or tissues from LRAT knockout models

• Antibody controls:

  • Isotype control (rabbit IgG with biotin conjugation)

  • No primary antibody control (to assess non-specific binding of detection reagents)

• Biotin-specific controls:

  • Endogenous biotin blocking control (especially important in biotin-rich tissues)

  • Streptavidin-only control (without biotin-conjugated antibody)

• Peptide competition control: Pre-incubation of antibody with immunizing peptide to verify specificity

These controls help distinguish specific LRAT detection from potential artifacts and validate experimental findings.

How can researchers distinguish between LRAT monomers and dimers in experimental systems?

Distinguishing between LRAT monomers (26 kDa) and dimers (50-54 kDa) requires careful consideration of sample preparation and analytical techniques:

• Sample preparation conditions:

  • Use of reducing agents (DTT or β-mercaptoethanol) in sample buffers disrupts disulfide bonds, favoring detection of the 26 kDa monomeric form

  • Non-reducing conditions preserve disulfide-linked dimers, allowing detection of the 50-54 kDa form

  • Native conditions maintain protein-protein interactions that stabilize dimers

• Analytical techniques:

  • SDS-PAGE with Western blotting using different sample buffer conditions

  • Native PAGE to preserve native protein-protein interactions

  • Size exclusion chromatography to separate monomers and dimers based on molecular size

  • Cross-linking experiments to stabilize transient interactions before analysis

• Data interpretation:

  • Compare band patterns under reducing vs. non-reducing conditions

  • Correlate molecular weight observations with functional activity

  • Consider the relative abundance of monomer vs. dimer forms in different subcellular fractions

This approach provides insights into the functional state of LRAT protein in experimental systems and may reveal important regulatory mechanisms.

What considerations should be made when investigating LRAT in retinal disease models?

When investigating LRAT in retinal disease models, several important considerations should guide experimental design:

• Disease relevance:

  • LRAT defects are linked to severe early-onset retinal dystrophy

  • Consider how LRAT dysfunction affects the visual cycle and retinoid metabolism

• Model selection:

  • Animal models with LRAT mutations or knockouts

  • Human patient-derived samples or iPSC-derived retinal cells

  • Retinal cell lines (e.g., Y79) that express LRAT

• Technical approaches:

  • Immunohistochemical localization to assess LRAT distribution in retinal layers

  • Western blot to quantify LRAT expression levels (monomer vs. dimer ratios)

  • Functional assays to measure retinyl ester formation activity

  • Gene expression analysis to investigate regulatory mechanisms

• Biomarker correlation:

  • Correlate LRAT expression/activity with visual function metrics

  • Assess relationship between LRAT levels and other components of the visual cycle

  • Determine progression markers for retinal degeneration in relation to LRAT status

This comprehensive approach facilitates mechanistic understanding of LRAT's role in retinal pathology and may identify therapeutic targets.

How does biotin conjugation impact antibody binding kinetics and epitope accessibility?

Biotin conjugation can influence antibody binding kinetics and epitope accessibility in several ways that researchers should consider:

Understanding these factors is essential for accurate interpretation of experimental results and may necessitate protocol adjustments when transitioning from unconjugated to biotin-conjugated LRAT antibodies.

How can researchers address non-specific binding when using biotin-conjugated LRAT antibody?

Non-specific binding is a common challenge when working with biotin-conjugated antibodies. To address this issue with biotin-conjugated LRAT antibody:

• Endogenous biotin blocking:

  • Pretreat samples with avidin followed by biotin blocking solutions

  • This prevents detection reagents from binding to endogenous biotin in tissues

• Buffer optimization:

  • Include blocking proteins like BSA (3-5%) in dilution buffers

  • Add 0.1-0.3% Triton X-100 or Tween-20 to reduce hydrophobic interactions

  • Consider adding 0.1-0.5M NaCl to reduce ionic interactions

• Secondary reagent considerations:

  • Use streptavidin conjugates with minimal batch-to-batch variation

  • Pre-absorb streptavidin conjugates against tissues of interest

  • Titrate streptavidin reagents to minimize background

• Sample-specific considerations:

  • When using this biotinylated antibody in tissues with cross-reacting endogenous immunoglobulins, dilute in buffers containing 2% normal serum from the same species as the tissue

  • Add 2-5% serum from the species providing the streptavidin conjugate

• Protocol modifications:

  • Include additional washing steps with increased detergent concentration

  • Reduce incubation temperature (4°C overnight instead of room temperature)

  • Decrease incubation time of detection reagents

These strategies can significantly improve signal-to-noise ratio and experimental reliability.

What are common pitfalls in data interpretation when studying LRAT using antibody-based techniques?

When studying LRAT using antibody-based techniques, researchers should be aware of several potential pitfalls in data interpretation:

Awareness of these potential pitfalls enables more rigorous experimental design and more accurate interpretation of LRAT research data.

How can researchers validate the specificity of biotin-conjugated LRAT antibody?

Validating the specificity of biotin-conjugated LRAT antibody is crucial for ensuring reliable research findings. Implement these validation strategies:

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide (recombinant LRAT protein 24-182AA)

  • Compare signal between blocked and unblocked antibody

  • Specific binding should be significantly reduced with peptide competition

• Genetic models:

  • Test antibody in LRAT-knockout or LRAT-overexpressing systems

  • Signal should correlate with genetic LRAT expression status

  • Use siRNA knockdown to create gradient expression levels for validation

• Orthogonal detection methods:

  • Compare antibody-based detection with mRNA expression (RT-PCR, RNA-seq)

  • Correlate protein detection with enzymatic activity assays

  • Use multiple antibodies targeting different LRAT epitopes

• Cross-reactivity assessment:

  • Test against recombinant proteins from the H-rev107 family

  • Evaluate specificity across multiple species if cross-species reactivity is claimed

  • Perform immunoprecipitation followed by mass spectrometry to identify all bound proteins

• Technical validation:

  • Compare staining/detection patterns between biotin-conjugated and unconjugated antibody versions

  • Evaluate signal in tissues/cells known to express or lack LRAT

  • Test different detection systems to confirm consistent results

Comprehensive validation ensures that experimental findings accurately reflect LRAT biology rather than technical artifacts.

How might biotin-conjugated LRAT antibody be utilized in multiplexed imaging applications?

Biotin-conjugated LRAT antibody offers significant potential for multiplexed imaging applications through these innovative approaches:

• Orthogonal detection systems:

  • Combine with directly fluorophore-labeled antibodies against other targets

  • Use streptavidin conjugated to spectrally distinct fluorophores or quantum dots

  • Implement sequential detection with antibody stripping between rounds

• Multi-omics integration:

  • Couple with RNA detection methods (RNAscope, FISH) for protein-transcript correlation

  • Combine with proximity ligation assays to detect LRAT protein interactions

  • Integrate with metabolic labeling to track retinyl ester production

• Advanced microscopy applications:

  • Super-resolution microscopy to resolve LRAT subcellular localization

  • Live-cell imaging using cell-permeable streptavidin conjugates

  • FRET-based approaches to detect LRAT-protein interactions

• Tissue and disease profiling:

  • Multiplex with markers of retinal cell types to map LRAT distribution

  • Compare normal vs. diseased tissue with parallel detection of disease markers

  • Developmental profiling with stage-specific markers

These approaches would provide unprecedented insights into LRAT's spatial relationships with other proteins and its role in retinal physiology and pathology.

What emerging technologies might enhance the utility of LRAT antibody detection systems?

Several emerging technologies hold promise for enhancing LRAT antibody detection systems:

• Advanced amplification methods:

  • Tyramide signal amplification (TSA) with biotin-conjugated LRAT antibody

  • DNA-barcoded antibodies for ultrasensitive digital counting

  • Proximity extension assays for highly specific detection

• Nanotechnology integration:

  • Plasmonic nanoparticles for enhanced optical detection

  • Magnetic nanoparticles for extraction and enrichment of LRAT-expressing cells

  • Nanobody-based detection systems with improved tissue penetration

• Microfluidic applications:

  • Single-cell LRAT protein quantification platforms

  • Automated immunostaining systems for improved reproducibility

  • Organ-on-chip models incorporating LRAT detection

• Computational advances:

  • AI-assisted image analysis for quantitative LRAT expression mapping

  • Machine learning algorithms to correlate LRAT patterns with disease phenotypes

  • Integrative multi-omics data analysis frameworks

• In vivo applications:

  • Development of non-invasive imaging for LRAT activity in animal models

  • Translational biomarkers for retinal diseases based on LRAT status

  • Therapeutic monitoring systems for interventions targeting the visual cycle

These technological advances will expand the utility of LRAT antibodies beyond current applications and enable new discoveries about LRAT biology and pathology.

How might LRAT detection contribute to understanding retinal disease mechanisms and developing therapeutics?

LRAT detection using biotin-conjugated antibodies and other advanced methods can significantly contribute to understanding retinal disease mechanisms and therapeutic development:

• Disease mechanism elucidation:

  • Mapping LRAT expression changes in progressive retinal degeneration

  • Identifying cell type-specific LRAT dysfunction in disease models

  • Understanding post-translational modifications of LRAT in pathological states

  • Correlating LRAT distribution with functional visual deficits

• Biomarker development:

  • Establishing LRAT expression patterns as diagnostic or prognostic indicators

  • Monitoring treatment response through LRAT function restoration

  • Identifying patient subgroups based on LRAT-related pathology patterns

  • Developing companion diagnostics for visual cycle-targeting therapies

• Therapeutic target validation:

  • Screening compounds that modulate LRAT expression or activity

  • Validating gene therapy approaches for LRAT-associated retinal dystrophies

  • Testing small molecule chaperones to rescue misfolded LRAT variants

  • Developing enzyme replacement strategies for LRAT deficiency

• Regenerative medicine applications:

  • Assessing LRAT functionality in stem cell-derived retinal pigment epithelium

  • Quality control for cell replacement therapies

  • Monitoring integration and function of transplanted cells

  • Validating tissue-engineered retinal constructs

These applications demonstrate how advanced LRAT detection methods, including biotin-conjugated antibodies, can accelerate both basic understanding of retinal biology and the development of targeted therapeutics for vision-threatening diseases.