NOTE: Revisions to “Preparationof Reagents” and “Assay Protocol”

Product Manual

L-Amino Acid Assay Kit (Fluorometric)
Catalog Number
MET-5055

100 assays

FOR RESEARCH USE ONLY
Not for use in diagnostic procedures

Introduction
Amino acids are organic compounds that contain amine (-NH2) and carboxyl (-COOH) functionalgroups, as well as a side-chain (R group) which confers uniqueness to each amino acid. The mainelements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, although other elements can befound in some amino acids. About 500 amino acids are known, however, only 20 are coded in thehuman genome. Amino acids are the monomers which are joined together to make short polymer
chains called peptides or longer chains called proteins. Non-protein amino acids play important roles in
the formation of biologically important molecules. For example, tryptophan is processed into theneurotransmitter serotonin, while tyrosine (and its precursor phenylalanine) is processed into
neurotransmitters dopamine, epinephrine and norepinephrine.
When consumed and absorbed by the human body, the standard amino acids are used to make proteins
and other molecules or are oxidized to urea and carbon dioxide to be used as a form of energy. Theoxidation pathway begins with transamidase removal of the amino group, and this group is thenprocessed through the urea cycle. The other transamidation product is a keto acid that is used for thecitric acid cycle. Through the process of gluconeogenesis, some amino acids can also be converted intoglucose. Out of the standard 20 amino acids, nine amino acids (His, Ile, Leu, Lys, Met, Phe, Thr, Trpand Val), are considered to be essential amino acids because the human body cannot make them fromother molecules in enough amounts needed for normal growth, so they must be obtained from foodsources.
Cell Biolabs’ L-Amino Acid Assay Kit is a simple fluorometric assay that measures the amount of total
free L-Amino Acids (except for Glycine) present in foods or biological samples in a 96-well microtiterplate format. Amino Acids in polypeptide chains (peptides and proteins) are not detected. Each kit
provides sufficient reagents to perform up to 100 assays, including blanks, L-Alanine standards andunknown samples. Sample L-Amino Acid concentrations are determined by comparison with a known
L-Alanine standard. The kit has a detection sensitivity limit of 6.25 µM L-Amino Acids.

Assay Principle
Cell Biolabs’ L-Amino Acid Assay Kit measures L-Amino Acids within food or biological samples.
An L-amino acid is oxidatively deaminated by L-Amino Acid oxidase into its corresponding α-ketoacid plus ammonia and hydrogen peroxide. The hydrogen peroxide is then detected with a highly
specific fluorometric probe. Horseradish peroxidase catalyzes the reaction between the probe andhydrogen peroxide, which bind in a 1:1 ratio. Samples and standards are read with a standard 96-wellfluorometric plate reader. Samples are compared to a known concentration of L-Alanine standard
within the 96-well microtiter plate format (Figure 1).

Figure 1. L-Amino Acid Assay Principle.

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Related Products
1.
2.
3.
4.
5.

MET-5056: Branched Chain Amino Acids (Colorimetric)
MET-5029: Pyruvate Assay Kit (Fluorometric)
MET-5070: Glycine Assay Kit (Fluorometric)
MET-5129: Lysine Assay Kit (Fluorometric)
STA-674: Glutamate Assay Kit (Fluorometric)

Kit Components
1. L-Alanine Standard (Part No. 50541C): One 30 µL tube at 100 mM.
2. 1X Assay Buffer (Part No. 50542A): One 25 mL bottle.
3. Fluorometric Probe (Part No. 50231C): One 50 μL amber tube.
4. HRP (Part No. 234402): One 100 μL tube at 100 U/mL in glycerol
5. L-Amino Acid Oxidase (Part No. 50544B): One 2.5 mL tube at 1 U/mL
Note: One unit is defined as the amount of enzyme that will oxidatively deaminate 1.0 µmole of L-leucine
per minute at pH 6.5 and 25°C.

Materials Not Supplied
1. Distilled or deionized water
2. 1X PBS
3. 96-well black or fluorescence-compatible microtiter plate

StorageUpon receipt, store the L-Alanine Standard, Fluorometric Probe, and HRP at -20ºC. The FluorometricProbe is light sensitive and must be stored accordingly. Avoid multiple freeze/thaw cycles. Store theL-Amino Acid Oxidase at 4°C (DO NOT FREEZE L-Amino Acid Oxidase). Store the 1X Assay
Buffer at room temperature.

Preparation of Reagents•Reaction Mix: Prepare a Reaction Mix by diluting the Fluorometric Probe 1:100, HRP 1:500, andL-Amino Acid Oxidase 1:30 in 1X Assay Buffer. For example, add 10 μL Fluorometric Probestock solution, 2 μL HRP stock solution, and 33.3 µL of L-Amino Acid Oxidase to 954.7 μL of 1X
Assay Buffer for a total of 1 mL. This Reaction Mix volume is enough for 20 assays. The Reaction
Mix is stable for 1 day at 4ºC.
Note: Prepare only enough for immediate use by scaling the above example proportionally.

Preparation of Samples•Tissue lysates: Sonicate or homogenize tissue sample in cold PBS or 1X Assay Buffer andcentrifuge at 10000 x g for 10 minutes at 4°C. Perform dilutions in 1X Assay Buffer.

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Cell lysates: Resuspend cells at 1-2 x 106 cells/mL in PBS or 1X Assay Buffer. Homogenize orsonicate the cells on ice. Centrifuge to remove debris. Cell lysates may be assayed undiluted or
diluted as necessary in 1X Assay Buffer.
Serum, plasma or urine: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The
supernatant may be assayed directly or diluted as necessary in 1X Assay Buffer.

Notes:• All samples should be assayed immediately or stored at -80°C for up to 1-2 months. Run propercontrols as necessary. Optimal experimental conditions for samples must be determined by theinvestigator. 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 the Fluorometric Probe is not stablein the presence of thiols (above 10 μM).

Preparation of Standard Curve
Prepare fresh L-Alanine standards before use by diluting in 1X Assay Buffer according to Table 2below.
Standard Tubes
1234567
8100 mM L-Alanine Solution (µL)
5250 of Tube #1
250 of Tube #2
250 of Tube #3
250 of Tube #4
250 of Tube #5
250 of Tube #601X Assay Buffer (µL)
1245
250
250
250
250
250
250250

L-Alanine (µM)
4002001005025
12.5
6.25
0Table 2. Preparation of L-Amino Acid Standards.

Assay Protocol
1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns andstandards, should be assayed in duplicate or triplicate.
2. Add 50 µL of each L-Alanine standard or unknown sample into wells of a 96-well microtiter plate.
3. Add 50 µL of Reaction Mix to each well. Mix the well contents thoroughly and incubate for 180
minutes at 37ºC protected from light.
Note: This assay is continuous (not terminated) and therefore may be measured at multiple time
points to follow the reaction kinetics.
4. 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.
5. Calculate the concentration of L-Amino Acids within samples by comparing the sample
fluorescence to the standard curve.
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Example of Results
The following figures demonstrate typical L-Amino Acid Assay Kit (Fluorometric) results. One should
use the data below for reference only. This data should not be used to interpret or calculate actual
sample results.

Figure 2: L-Alanine standard curve.

Figure 3: L-Amino Acid detection in human serum using the L-Amino Acid Assay Kit (Fluorometric).

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References
1.
2.
3.
4.
5.
6.
7.
8.

Wagner I, Musso H (1983). Angew. Chem. Int. 22: 816–828.
Votyakova TV, and Reynolds IJ (2001) Neurochem. 79:266.
Sakami W, Harrington H (1963). Ann. Rev Biochem. 32: 355–98.
Brosnan JT (2000). J. Nutrit. 130 (4S Suppl): 988S–90S.
Young VR, Ajami AM (2001). J. Nutrit. 131 (9 Suppl): 2449S–59S.
Young VR (1994). J.Nutrit. 124 (8 Suppl): 1517S–1523S.
Fürst P, Stehle P (2004). J. Nutrit. 134 (6 Suppl): 1558S–1565S.
Reeds PJ (2000). J.Nutrit. 130 (7 Suppl): 1835S–40S.

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 OR
WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CELL BIOLABS’s 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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