Product Manual

Tryptophan Assay Kit
Catalog Number
MET-5177

100 assays

FOR RESEARCH USE ONLY
Not for use in diagnostic procedures

Introduction
Tryptophan is an essential amino acid (obtained from the diet) that is used in protein biosynthesis.
Tryptophan is a polar molecule since it contains an α-amino group, an α-carboxylic acid group, and aside chain indole. Tryptophan is a precursor for the synthesis of the neurotransmitter serotonin, thehormone melatonin, and vitamin B3. Serum albumin (such as BSA) binds tryptophan in a 1:1 ratio.
Tryptophan is sold in the United States and the United Kingdom as a dietary supplement to treat
depression, anxiety, and insomnia. Tryptophan is also sold under prescription in some European
countries to treat major depression.
Cell Biolabs’ Tryptophan Assay Kit is a simple assay for measuring tryptophan levels in biologicalsamples. Tryptophan levels may be quantified in serum or plasma following a special pretreatment
protocol (see Preparation of Samples section). The kit has a detection sensitivity limit of 1.56 µM
tryptophan. Each kit provides sufficient reagents to perform up to 100 assays*, including standard
curve and unknown samples.
*Note: Each sample replicate requires 2 assays, one treated with Tryptophan Oxidase (+TO) and
one without (-TO). Tryptophan level is calculated from the difference in RFU readings from the 2wells.

Assay Principle
The Tryptophan Assay Kit is a sensitive quantitative fluorometric assay for tryptophan. Tryptophan
Oxidase converts tryptophan to 2-imino-3(indol-3-yl) propanoate and hydrogen peroxide (H2O2). The
H2O2 is then detected with a highly specific fluorometric probe. Horseradish peroxidase catalyzes thereaction between the probe and hydrogen peroxide, which bind in a 1:1 ratio. Samples and standards
are incubated for 60 minutes and then read with a standard 96-well fluorometric plate reader. Samples
are compared to a known concentration of tryptophan standard within the 96-well microtiter plateformat.

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Related Products
1. MET-5054: L-Amino Acid Assay Kit
2. MET-5056: Branched Chain Amino Acid Assay Kit
3. MET-5071: Taurine Assay Kit
4. MET-5073: Tyrosine Assay Kit
5. MET-5129: Lysine Assay Kit (Fluorometric)

Kit Components
1. L-Tryptophan Standard (Part No. 51771C): One 100 L vial at 10 mM.
2. 10X Assay Buffer (Part No. 51772A): One 30 mL bottle
3. Tryptophan Oxidase (Part No. 51773D): One 500 L vial.
4. Fluorometric Probe (Part No. 50231C): One 50 µL tube in DMSO.
5. HRP (Part No. 234402): One 100 μL tube of a 100 U/mL solution in glycerol.

Materials Not Supplied
1. Fluorescence microplate reader capable of reading excitation in the 530-570 nm range andemission in the 590-600 nm range
2. 6.12 M Trichloroacetic Acid (CAS # 76-03-9)
3. 6 M Sodium Bicarbonate (CAS # 144-55-8)
4. 10 kD cutoff Centrifugal Filter Device

StorageUpon receipt, store the Tryptophan Oxidase at -80°C. Store all other components at -20ºC. Avoid
multiple freeze/thaw cycles. The Fluorometric Probe is light sensitive and must be stored accordingly.

Preparation of Reagents
Note: All reagents must be brought to room temperature prior to use.
• 1X Assay Buffer: Dilute the stock 10X Assay Buffer 1:10 with deionized water for a 1X solution.
Stir or vortex to homogeneity.

3• Reaction Mix: Prepare mixture according to the table below. Scale appropriately as needed andprepare only enough for immediate use.
Component

Reaction Mix(20 assays)

Fluorometric Probe

10 µL

HRP

2 µL

Tryptophan Oxidase

100 µL1X Assay Buffer

888 µL

Total

1000 µL• Negative Control Mix: Prepare mixture according to the table below. Scale appropriately as neededand prepare only enough for immediate use.
Component

Negative
Control Mix(20 assays)

Fluorometric Probe

10 µL

HRP

2 µL

1X Assay Buffer

988 µL

Total

1000 µL

Preparation of Samples•

Cell culture supernatants: To remove insoluble particles, centrifuge at 10,000 x g for 10 min at
4°C. Collect the supernatant and filter the solution with a 10 kDa spin filter to deproteinate thesample. Collect flow through. The flow through can be assayed directly or diluted as necessary into
1X Assay Buffer. Prepare the Tryptophan standard curve in the same non-conditioned
deproteinated media.
Note: Maintain pH between 7 and 8 for optimal working conditions as the Fluorometric Probe isunstable at high pH (>8.5).

•Cell lysates: Resuspend cells in 1X Assay Buffer. Homogenize or sonicate the cells on ice.
Centrifuge 10,000 x g for 10 minutes at 4°C to remove debris. Collect the supernatant and filter thesolution with a 10kD cutoff Centrifugal Filter Device to deproteinate the sample. Collect flow
through. The flow through may be assayed undiluted or diluted as necessary into 1X Assay Buffer.

•Tissue lysates: Sonicate or homogenize tissue sample in 1X Assay Buffer and centrifuge at 10,000x g for 10 minutes at 4°C. Collect the supernatant and filter the solution with a 10kD cutoff

4Centrifugal Filter Device to deproteinate the sample. Collect flow through. The flow through may
be assayed directly or diluted as necessary into 1X Assay Buffer.
•Serum or Plasmao Free Tryptophan: Deproteinate human serum by loading 500 µL into a 10kD cutoff
Centrifugal Filter Device. Spin 14000 xg for 10-30 minutes and collect the flowthrough. Store solution at -20°C until ready to test (dilute into 1X Assay Buffer asnecessary).
o Total Tryptophan (Tryptophan needs to be peripherally extricated from serum albumin):
Dilute 6.12 M trichloroacetic acid (TCA) 1:3 to 2.04 M in distilled water. Add 100 µLof 2.04 M TCA to 400 µL of serum or plasma in a microcentrifuge tube and mix bypipetting up and down. Centrifuge the sample at 13000xg for 5 minutes at 4°C. Transfer
380 µL of supernatant to a fresh microcentrifuge tube. Neutralize by slowly adding 125µL of 6 M sodium bicarbonate (NaHCO3) (Important Note: add NaHCO3 DROPWISE
to avoid bubbling). Mix the solution gently by pipetting up and down. Store solution at-20°C until ready to test (dilute into 1X Assay Buffer as necessary).

•Urine: To remove insoluble particles, centrifuge at 10000 x g for 10 min at 4°C. Collect thesupernatant and filter the solution with a 10kD cutoff Centrifugal Filter Device to deproteinate thesample. Collect flow through. The flow through can be assayed directly or diluted as necessary into
1X Assay Buffer.
Notes:• All samples should be assayed immediately or stored at -80°C for up to 1-2 months. Run
proper controls as necessary. Optimal experimental conditions for samples must be
determined by the investigator. Always run a standard curve with samples.
• Samples with NADH concentrations above 10 μM and glutathione concentrations above 50 μM
will oxidize the Fluorometric Probe and could result in erroneous readings. To minimize thisinterference, it is recommended that superoxide dismutase (SOD) be added to the reaction at a
final concentration of 40 U/mL (Votyakova and Reynolds, Ref. 2).
• Avoid samples containing DTT or β-mercaptoethanol since Resorufin is not stable in thepresence of thiols (above 10 μM).

Preparation of Standard Curve
Prepare fresh Tryptophan standards before use by diluting in distilled water according to Table 1below.

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10 mM L-Tryptophan
SolutionStandard Tubes(µL)
Distilled Water (µL)
15495
2250 of Tube #1250
3250 of Tube #2250
4250 of Tube #3250
5250 of Tube #4250
6250 of Tube #5250
7250 of Tube #6250
80250
Table 1. Preparation of L-Tryptophan Standards

L-Tryptophan(µM)
100
5025
12.5
6.25
3.13
1.56
0Assay Protocol
1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns andstandards, should be assayed in duplicate or triplicate.
Note: Each unknown sample replicate requires two paired wells, one to be treated with Tryptophan
Oxidase (+TO) and one without the enzyme (-TO) to measure endogenous background.
2. Add 50 μL of each Tryptophan Standard or unknown sample into wells of a 96-well microtiterplate.
3. Add 50 μL of Reaction Mix (+TO) to the standards and to one half of the paired sample wells, andmix the well contents thoroughly.
4. Add 50 µL of Negative Control Mix (-TO) to the other half of the paired sample wells.
5. Mix the well contents thoroughly and incubate for 60 minutes at room temperature protected fromlight.
Note: This assay is continuous (not terminated) and therefore may be measured at multiple time
points to follow the reaction kinetics.
6. Read the plate with a fluorescence microplate reader equipped for excitation in the 530-570 nm
range and for emission in the 590-600 nm range.

Calculation of Results
1. Determine the average relative fluorescence unit (RFU) values for each sample, control, andstandard.
3. Subtract the average zero standard value from itself and all standard values.
4. Graph the standard curve (see Figure 1).
5. Subtract the sample well values without Tryptophan Oxidase (-TO) from the sample well values
containing Tryptophan Oxidase (+TO) to obtain the difference. The fluorescence difference is due tothe Tryptophan Oxidase activity:

Net RFU = RFU(+TO) – RFU(-TO)

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6. Compare the Net RFU of each sample to the standard curve to determine and extrapolate thequantity of Tryptophan present in the sample. Only use values within the range of the standardcurve.
7. To calculate the final tryptophan concentration in the sample, take into account any prior dilutions
performed (for example in the total tryptophan plasma/serum protocol, sample is diluted 6.6-fold)

Example of Results
The following figures demonstrate typical Tryptophan Assay results. One should use the data below
for reference only. This data should not be used to interpret actual results.

Figure 1. Tryptophan Standard Curve.

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Figure 2. Detection of total or free tryptophan in human serum or urine. Human serum or urine
was deproteinated according to the preparation of samples section above.

References
1. Cunningham VJ, Hay L, and Stoner HB (1975) Biochem J. 146: 653-658.
2. Votyakova TV, and Reynolds IJ (2001) Neurochem. 79:266-277.
3. Slominski A, Semak I, Pisarchik A, Sweatman T, Szczesniewski A, Wortsman J (2002). FEBS
Letters. 511: 102–6.
4. Schaechter JD, Wurtman RJ (1990) Brain Research. 532: 203–10.
5. Wurtman RJ, Anton-Tay F (1969) Recent Progress in Hormone Research. 25: 493–522
6. Ikeda M, Tsuji H, Nakamura S, Ichiyama A, Nishizuka Y, Hayaishi O (1965). J. Biol. Chem. 240:1395–401
7. Soh NL, Walter GT (2011). Acta Neuropsychiatrica. 23: 1601–5215
8. Wurtman RJ, Hefti F, Melamed E (1980). Pharmacological Reviews. 32: 315–35

Warranty
These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used inaccordance with their instructions. THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSEDWARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY ORWARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CELL BIOLABS’ sole obligation and purchaser’s
exclusive remedy for breach of this warranty shall be, at the option of CELL BIOLABS, to repair or replace the products.
In no event shall CELL BIOLABS be liable for any proximate, incidental or consequential damages in connection with theproducts.

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Contact Information
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5628 Copley Drive
San Diego, CA 92111
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E-mail: tech@cellbiolabs.com
www.cellbiolabs.com©2024: Cell Biolabs, Inc. - All rights reserved. No part of these works may be reproduced in any form without permissions
in writing.

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