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

Product Manual

L-Amino Acid Assay Kit (Colorimetric)
Catalog Number
MET-5054

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, but only 20 are coded in the humangenome. 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 theformation 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 colorimetric assay that measures the total amount of
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 62.5 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 colorimetric 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-wellcolorimetric 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 Acid Assay Kit (Colorimetric)
MET-5068: Folic Acid Elisa Kit (Colorimetric)
MET-5125: Pyruvate Assay Kit (Colorimetric)
MET-5027: Homocitrulline/ Citrulline Assay Kit (Colorimetric)
MET-5070: Glycine 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. Colorimetric Probe (Part No. 50122C): 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 170 µL tube at 32 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 PBS3. Standard 96-well clear microtiter plate and/or clear cell culture microplate

StorageUpon receipt, store the L-Alanine Standard, Colorimetric Probe, and HRP at -20ºC. The Colorimetric
Probe 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 Colorimetric Probe 1:100, HRP 1:500, and LAmino Acid Oxidase 1:30 in 1X Assay Buffer. For example, add 10 μL Colorimetric Probe stocksolution, 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 isstable 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 Colorimetric 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 Colorimetric 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 2.
Standard Tubes
123456

100 mM L-Alanine Solution (µL) 1X Assay Buffer (µL)
5495
250 of Tube #1250
250 of Tube #2250
250 of Tube #3250
250 of Tube #4
2500250

L-Alanine (µM)
1000
500
250
125
62.5
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 spectrophotometric microplate reader in the 540-570 nm range.
5. Calculate the concentration of L-Amino Acids within samples by comparing the sample OD to thestandard curve.

4Example of Results
The following figures demonstrate typical L-Amino Acid Assay Kit (Colorimetric) 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 (Colorimetric).

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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.

Recent Product Citation
Okamoto, E. et al. (2023). Regulation of Paracellular Fluxes of Amino Acids by Claudin-8 in NormalMouse Intestinal MCE301 Cells. Nutrients. 15(6):1346. doi: 10.3390/nu15061346.

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no event shall CELL BIOLABS be liable for any proximate, incidental or consequential damages in connection with theproducts.

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