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

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.

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).

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.

References Using Mediomics’ Bridge-It L-Tryptophan Fluorescence Assay Kit

1. The Effect of Branched-Chain Amino Acids, Citrulline, and Arginine on High-Intensity Interval Performance in Young Swimmers. Hsueh CF, Wu HJ, Tsai TS, Wu CL, Chang CK. Nutrients. 2018 Dec 14;10(12). pii: E1979.

2. Profiling targetable immune checkpoints in osteosarcoma. McEachron TA, Triche TJ, Sorenson L, Parham DM, Carpten JD. Oncoimmunology. 2018 Sep 11;7(12):e1475873.

3. Branched-chain amino acids and arginine improve physical but not skill performance in two consecutive days of exercise. Lin YT, Chiu MS, Chang CK. Sci Sport. 2017 Dec; 32(6):e221-e228.

4. Involvement of Host Defense Mechanisms against Toxoplasma gondii Infection in Anhedonic and Despair-Like Behaviors in Mice. Mahmoud ME, Fereig R, Nishikawa Y. Infect Immun. 2017 Mar 23;85(4). pii: e00007-17.

5. Quantitative Protein Profiling of Chlamydia trachomatis Growth Forms Reveals Defense Strategies Against Tryptophan Starvation. Østergaard O, Follmann F, Olsen AW, Heegaard NH, Andersen P, Rosenkrands I. Mol Cell Proteomics. 2016 Dec;15(12):3540-3550.

6. Challenges in the Discovery of Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors. Röhrig UF, Majjigapu SR, Vogel P, Zoete V, Michielin O. J Med Chem. 2015 Dec 24;58(24):9421-37.

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

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 : 

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

1 file(s) 206.92 KB

SDS :

Tryptophan Fluorescence Assay Kit SDS

1 file(s) 200.18 KB