Bridge-It® L-Tryptophan Fluorescence Assay Kit, 96-well format

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Description

Bridge-It® L-Tryptophan Fluorescence Assay Kit, 96-well format

Application:

  • Quantification of Tryptophan levels in biological fluids, cell culture and fermentation medium, and extracts of tissues and cells.

  • Measurement of total tryptophan and free tryptophan

  • High throughput screening for drug discovery (please contact us for bulk discount pricing)

  • Diet and nutrition, mental health, and immunology studies

 

Features:

  • Easy: Mix test sample or standard with assay solution and incubate at ~25°C

  • Fast: Read fluorescent signal after 30-minute incubation (Reader settings: excitation 485 nm; emission 665 nm)

  • Sensitive: Assay measures L-tryptophan levels as low as ~0.078 µM 

  • Flexible: Method is adaptable to high-throughput screening formats

  • Specific: No cross reactivity with other standard amino acids or Kynurenine

  • Highly Reproducible : Measure total tryptophan in serum and plasma samples with high reproducibility and accuracy


 

Background Information on L-Tryptophan and the Bridge-It Fluorescence Assay

L-Tryptophan L-tryptophan (tryptophan) is one of eight essential amino acids that must be obtained from the diet. Tryptophan serves as a key building block for synthesis of proteins and as a precursor for various brain neurotransmitters including serotonin. Tryptophan is the only recognized precursor that can be converted into serotonin by the body. Serotonin promotes feelings of well-being and calm and thereby helps to counterbalance the physiological effects of brain dopamine and the nor-adrenaline circuits which encourage fear, anger, tension, aggression, obsessive-compulsive actions, over-eating (especially of carbohydrates), migraine headache, depression, and sleep disturbances. Melatonin, a metabolite of serotonin, is a sleep promoting natural hormone made by the pineal gland. In addition, tryptophan is a key precursor for niacin (vitamin B3), a vitamin that is essential for normal respiration, metabolism, and synthesis of sex hormones. Because tryptophan plays such a critical role in the proper balancing of metabolism, mood and sleep patterns, insufficient dietary availability of this essential amino acid can lead to serious adverse consequences.

 

Bridge-It® L-Tryptophan Fluorescence Assay

The Bridge-It® tryptophan fluorescence assay is based on the activity of tryptophan repressor protein (TrpR), a bacterial DNA-binding protein. TrpR protein binds to its DNA-binding site in tryptophan-dependent fashion.

The central feature of this assay design is the TrpR-dependent association of two fluorochrome-labeled DNA half-fragments (one labeled with fluorescein and the other labeled with Oyster® 645 fluophore. Each fragment contains about one-half of the TrpR protein DNA-binding site. In the presence of L-tryptophan an increase in fluorescence signal can be detected as a result of the tryptophan-dependent association of the labeled DNA half-fragments. Tryptophan is readily detectable using the Bridge-It® tryptophan fluorescence assay in various types of test samples including bacterial growth medium, brain extract, yeast extract, as well as in human serum and urine. The range of the assay is 0.078 µM – 20 µM and the minimum tryptophan detection level is ~0.078 µM. The assay is highly specific for measuring tryptophan. No significant TrpR protein binding activity was observed using the assay when L-tryptophan was replaced with each of nineteen (19) other L-amino acids (up to 100 µM) or D-tryptophan, serotonin, and the tryptophan precursor 5’HTP (up to 20 µM).

Tryptophan_Std_96

The Bridge-It® L-tryptophan fluorescence assay is performed using the 96-well or 384-well microplate format and is, therefore, ideally suited for the rapid, simultaneous measurement of tryptophan in large numbers of test samples.

 

Pricing: The 50-well kit is a one-time trial purchase. For bulk pricing options, please contact us. For any other questions or comments, please do not hesitate to contact us. We are always happy to help!

 

For information on the assay principle please see the Bridge-It Assay Technology platform page or download the protocol. This product is protected by patents and pending patents.


Selected Customer Publications Using Mediomics’ Bridge-It L-Tryptophan Fluorescence Assay Kit

1. Röhrig UF, Majjigapu SR, Vogel P, Zoete V, Michielin O. Challenges in the Discovery of Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors.J. Med. Chem. 2015 May 13.

2. Chang CK, Chang Chien KM, Chang JH, Huang MH, Liang YC, Liu TH. Branched-Chain Amino Acids and Arginine Improve Performance in Two Consecutive Days of Simulated Handball Games in Male and Female Athletes: A Randomized Trial. PLOS One. 2015 March 24.

— “Plasma free tryptophan concentration was analyzed with a fluorescence assay according to manufacturer’s recommendation (Bridge-It, Mediomics, St. Louis, MO, USA).”

3. Piesiewicz A, Kedzierska U, Turkowska E, Adamska I, Majewski PM Seasonal postembryonic maturation of the diurnal rhythm of serotonin in the chicken pineal gland. Chronobiol Int. 2014 Sep 15.

— “…duplicate 10 µl aliquots were taken to measure TRP concentration with a Bridge-It L-tryptophan Fluorescence Assay Kit (Mediomics, St. Louis, MO).

4. Urata Y, et al. IL-1β Increases Expression of Tryptophan 2,3-dioxygenase and Stimulates Tryptophan Catabolism in Endometrioma Stromal Cells. AJRI, 26 Jun 2014.

— “Concentration of tryptophan in conditioned media was measured using Bridge-It L-Tryptophan Fluorescence Assay (Mediomics, St. Louis, MO, USA) according to the manufacturer’s protocol.”

5. Piesiewicz A, Kedzierska U, Adamska I, Usarek M, Zeman M, Skwarlo-Sonta K, Majewski PM. Pineal arylalkylamine N-acetyltransferase (Aanat) gene expression as a target of inflammatory mediators in the chicken. Gen Comp Endocrinol. 2012 Nov 1;179(2):143-51.

— “Pineal glands were sonicated (Bandelin electronic) in 25 μl of ice-cold sodium phosphate buffer (50 mM, pH 6.8) and duplicate 10 μl aliquots were taken to measure TRP concentration using a Bridge-It l -tryptophan Fluorescence Assay Kit (Mediomics).

6. Bacchus W, Lang M, El-Baba MD, Weber W, Stelling J, Fussenegger M. Synthetic two-way communication between mammalian cells. Nat Biotechnol. 2012 Oct;30(10):991-6.

— “L-tryptophan levels in the medium and serum were profiled using a fluorescence-based assay kit (Mediomics, St. Louis, MO, USA, cat. no. 1-1-1001B).”

7. Meininger D, Zalameda L, Liu Y, Stepan LP, Borges L, McCarter JD, Sutherland CL. Purification and kinetic characterization of human indoleamine 2,3-dioxygenases 1 and 2 (IDO1 and IDO2) and discovery of selective IDO1 inhibitors. Biochim Biophys Acta. 2011 Dec;1814(12):1947-54.

— “…were determined by measuring the production of the kynurenine product by kynurenine fluorescence or by the consumption of the Trp substrate using the Bridge-IT tryptophan fluorescence assay (Mediomics, St. Louis, MO).”

8. Piesiewicz A, Podobas E, Kedzierska U, Joachimiak E, Markowska M, Majewski P, Skwarlo-Sonta K. Season-Related Differences in the Biosynthetic Activity of the Neonatal Chicken Pineal Gland. The open Ornithology journal, 2010, 3, 134-140.

— “L-tryptophan was measured in duplicate pineal sonicate samples using a Bridge-It L-Tryptophan Fluorescence Assay Kit (Mediomics), according to the manufacturer’s protocol.”

 


  1. Bridge-It® is a registered trademark of Mediomics, LLC, St. Louis, Missouri, U.S.A. Mediomics has a worldwide, exclusive license for this assay platform from Saint Louis University, St. Louis, Missouri.

  2. The Bridge-It® tryptophan fluorescence assay is intended for laboratory research use only. This product is not approved by the U.S. Government or by the government of any other country of the world for use in disease diagnosis or treatment of humans or animals.

  3. Oyster® is a registered trademark of Denovo Biolabels, GmbH, Munster, Germany.

  4. Flownamics® Analytical Instruments, Inc., Madison, Wisconsin, is the authorized U.S. distributor for Oyster® dyes.

Catalog #: 1-1-1001A (50 measurements) , 1-1-1001B (96 measurements)

Protocol : 

SDS :