Miraculin Antibody

What is the source and basic structure of Miraculin?

Miraculin is a glycoprotein derived from the berries of Synsepalum dulcificum, also known as miracle fruit or previously classified as Richadella dulcifica. The protein has a UniProt ID of P13087 and consists of amino acids 30-220 in its recombinant form commonly used for antibody production . Miraculin functions by binding to sweet receptor cells on the tongue, inhibiting the perception of sour tastes by the brain, effectively converting sour flavors into sweet ones without being inherently sweet itself .

How is the Miraculin polyclonal antibody produced?

The production of Miraculin polyclonal antibody follows a well-structured immunological process:

• Repeated immunization of rabbits using recombinant Synsepalum dulcificum miraculin (30-220aa) until optimal antibody titer is achieved

• Collection of blood from the immunized rabbits

• Meticulous purification of antibodies from serum using protein A/G chromatography

• Extensive functional assessment through ELISA and Western Blot applications

• Confirmation of specific reactivity with Synsepalum dulcificum miraculin

The resulting antibody is stored in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative .

What are the validated applications for Miraculin antibody?

The Miraculin antibody has been extensively validated for the following research applications:

The antibody shows specific reactivity against Synsepalum dulcificum miraculin and is supplied as 50μl of purified IgG antibody .

How can Miraculin antibody be utilized in transgenic plant research?

Researchers have successfully expressed functional miraculin in transgenic tomato plants, achieving high and genetically stable expression confirmed through Western blot analysis and enzyme-linked immunosorbent assay (ELISA) . When designing experiments to detect miraculin in transgenic plants:

• Extract total protein from plant tissues using appropriate extraction buffers

• Separate proteins via SDS-PAGE and transfer to PVDF or nitrocellulose membranes

• Block membranes with 5% non-fat dry milk or BSA solution

• Incubate with Miraculin antibody (primary antibody) at appropriate dilution

• Wash and add species-specific HRP-conjugated secondary antibody

• Develop using chemiluminescent substrate and image using suitable detection system

Recombinant miraculin has been shown to accumulate to levels up to 102.5 μg/g fresh weight in leaves and 90.7 μg/g fresh weight in tomato fruits, with preserved sweetness-inducing activity comparable to native miraculin .

What methodologies are recommended for optimizing Miraculin antibody specificity in complex samples?

To optimize specificity when working with Miraculin antibody in complex biological samples:

• Pre-absorption technique: Incubate the antibody with recombinant Miraculin protein (available as positive control) before use to pre-absorb non-specific binding sites

• Cross-reactivity testing: Validate against related plant proteins to ensure specificity

• Gradient dilution series: Perform titration experiments to determine optimal antibody concentration that maximizes specific signal while minimizing background

• Extended blocking: Increase blocking time or use alternative blocking agents (e.g., fish gelatin) for samples with high background

• Addition of detergents: Include 0.1-0.3% Tween-20 in washing and antibody incubation buffers to reduce non-specific interactions

These optimization approaches are particularly important when distinguishing between native and recombinant miraculin in experimental systems .

What controls should be included when using Miraculin antibody in immunoassays?

A robust experimental design using Miraculin antibody should include the following controls:

• Positive control: Use the supplied recombinant immunogen protein (200 μg) to confirm antibody reactivity

• Negative control: Include samples from non-miraculin expressing plants or tissues

• Isotype control: Use non-specific rabbit IgG at the same concentration to identify background binding

• Secondary antibody control: Omit primary antibody to detect non-specific binding of secondary antibody

• Blocking peptide control: Pre-incubate antibody with excess miraculin peptide to confirm signal specificity

For quantitative assays, include a standard curve generated using purified recombinant miraculin protein at known concentrations to enable accurate quantification of target protein levels.

How should storage and handling protocols be optimized for long-term Miraculin antibody stability?

To maintain optimal Miraculin antibody performance over extended periods:

• Upon receipt, aliquot the antibody in smaller volumes to avoid repeated freeze-thaw cycles

• Store at -20°C or preferably -80°C for long-term stability

• Avoid more than 3-5 freeze-thaw cycles, as this can lead to protein denaturation and loss of activity

• When thawing, allow the antibody to reach room temperature gradually before use

• Centrifuge briefly after thawing to collect all liquid at the bottom of the tube

• Consider adding carrier proteins such as BSA (0.1-1%) to diluted antibody solutions to enhance stability

• For working dilutions, store at 4°C if using within 1-2 weeks, or re-freeze aliquots for longer storage

Proper storage and handling are critical for maintaining the antibody's specific reactivity with Synsepalum dulcificum miraculin over time.

How is Miraculin being investigated in clinical research settings?

Recent clinical research has explored the potential therapeutic applications of miraculin-based supplements, particularly for cancer patients experiencing taste disorders:

• A triple-blind, randomized, placebo-controlled clinical trial (CLINMIR Protocol) evaluated the effects of dried miracle berries (DMB) containing miraculin on taste perception and nutritional status in malnourished cancer patients undergoing active treatment

• The study design involved three treatment arms:

  • Standard dose: 150 mg DMB (equivalent to 2.8 mg miraculin) + 150 mg freeze-dried strawberries

  • High dose: 300 mg DMB (equivalent to 5.6 mg miraculin)

  • Placebo: 300 mg freeze-dried strawberries

• Patients consumed orodispersible tablets five minutes before each main meal over a three-month period

• Taste perception was objectively measured using electrogustometry, which quantifies taste threshold via electrical stimulation

Results showed promising effects on taste perception, suggesting miraculin's potential role in managing taste disorders in oncology patients and potentially improving nutritional outcomes .

What methodologies are used to study Miraculin's impact on oral microbiome?

Research has investigated miraculin's effects on the oral microbiome of cancer patients using the following methodological approaches:

• Collection of oral microbiome samples before and after miraculin supplementation

• DNA extraction and 16S rRNA gene sequencing to identify bacterial species

• Bioinformatic analysis to assess microbial diversity and abundance

• Statistical comparison between treatment groups (standard dose DMB, high dose DMB, and placebo)

Three bacterial species were found to dominate the oral microbiome of cancer patients: Streptococcus pneumoniae, Streptococcus thermophilus, and Veillonella parvula . Changes in the oral microbiome composition following regular DMB consumption may contribute to maintaining appropriate immune responses in these patients, though further research is needed to fully elucidate these mechanisms .

How can researchers address cross-reactivity issues when using Miraculin antibody?

When encountering cross-reactivity issues with Miraculin antibody:

• Increase antibody specificity:

  • Use higher dilutions of primary antibody

  • Extend washing steps in immunoassays

  • Add 0.1-0.5% non-ionic detergents to washing buffers

• Epitope mapping:

  • Determine specific epitopes recognized by the antibody

  • Use peptide competition assays to identify non-specific binding

• Pre-absorption protocol:

  • Incubate antibody with tissue/protein lysates from non-target species

  • Remove cross-reactive antibodies before use in critical applications

• Alternative detection methods:

  • Consider using mass spectrometry for definitive protein identification

  • Complement antibody-based detection with nucleic acid-based methods

The polyclonal nature of the Miraculin antibody means it recognizes multiple epitopes of the target protein, which can occasionally lead to cross-reactivity with structurally similar proteins .

What are the critical parameters for quantifying Miraculin expression in transgenic systems?

When quantifying miraculin expression in transgenic systems, researchers should consider these critical parameters:

Researchers have successfully quantified miraculin in transgenic tomato plants at levels reaching 102.5 μg/g fresh weight in leaves and 90.7 μg/g fresh weight in fruits . Proper quantification is essential for correlating expression levels with functional activity and determining optimal dosages for potential therapeutic applications.

What emerging research areas are utilizing Miraculin antibody technology?

Several promising research directions involve Miraculin antibody:

• Alternative expression systems: Beyond tomato plants, researchers are exploring other plant-based and microbial expression systems for higher-yield miraculin production

• Structural biology applications: Using antibodies to crystallize and determine high-resolution structures of miraculin to better understand its taste-modifying mechanism

• Biomarker development: Potential use of anti-miraculin antibodies in developing detection systems for quality control of miraculin-containing products

• Clinical applications expansion: Building on existing clinical trials to explore miraculin's potential benefits for patients with various taste disorders beyond cancer-related dysgeusia

• Microbiome-taste interaction studies: Further investigation into how miraculin affects the oral microbiome and whether these changes contribute to taste perception alterations

The unique properties of miraculin continue to attract research interest across multiple disciplines, from agricultural biotechnology to clinical nutrition and taste physiology .

How might computational approaches enhance Miraculin antibody design and applications?

Computational methods are increasingly valuable for advancing Miraculin antibody research:

• Epitope prediction: In silico analysis to identify immunogenic regions of miraculin for more targeted antibody development

• Molecular docking simulations: Modeling antibody-antigen interactions to predict binding affinities and optimize specificity

• Structural homology modeling: Predicting three-dimensional structures of miraculin variants to understand functional differences

• Machine learning applications: Using AI to analyze patterns in antibody binding data and predict cross-reactivity

• Systems biology integration: Combining antibody-based detection data with transcriptomic and metabolomic analyses for comprehensive understanding of miraculin biology

These computational approaches can help researchers design more specific antibodies, predict potential cross-reactivity issues, and develop more efficient detection methods for both native and recombinant miraculin in various experimental systems.