Product Manual

Lysine Assay Kit (Fluorometric)
Catalog Number
MET-5129

100 assays

FOR RESEARCH USE ONLY
Not for use in diagnostic procedures

Introduction
Lysine is an amino acid containing amine (-NH2) and carboxyl (-COOH) functional groups, as well as alysyl group side chain ((CH2) 4NH2). Lysine is an essential amino acid since it is not synthesized in thebody and therefore must be obtained from food sources.
Lysine is one of 22 natural amino acids that serve as fundamental building blocks for the production ofproteins. Lysine also plays a part in protein structure: its side chain contains a positively charged groupon one end connected to a long hydrophobic carbon tail. Because of these properties, lysine isconsidered relatively amphipathic and can be found either buried within the core of a protein or insolvent channels as well as on the exterior of proteins, where it can interact with the aqueousenvironment. Lysine can also aid in protein stabilization since its ε-amino group forms hydrogen bonds,
salt bridges, and covalent interactions to form a Schiff base. In addition, lysine is involved in
epigenetic regulation by means of direct histone modification. There are several covalent histone
modifications, often involving lysine residues located in the extending tail of histones. These
modifications effect gene regulation, in which genes can be either activated or repressed. Lysine has
also been shown to be involved in structural proteins of connective tissues, calcium homeostasis, andfatty acid metabolism. Lysine has been shown to play a role in the crosslinking between the threehelical polypeptides of collagen, resulting in collagen stabilization and increased tensile strength.
Lysine has also been suggested to play a role in calcium intestinal absorption and renal retention, andtherefore might be involved in calcium homeostasis. Finally, lysine is a known precursor of carnitine, amolecule that transports fatty acids to the mitochondria to be oxidized resulting in energy release.
Cell Biolabs’ Lysine Assay Kit is a simple fluorometric assay that measures the amount of free lysine
present in foods or biological samples in a 96-well microtiter plate format. Lysine found in polypeptide
chains (peptides and proteins) are not detected. Each kit provides sufficient reagents to perform up to100 assays, including blanks, lysine standards and unknown samples. Sample lysine concentrations aredetermined by comparison with a known lysine standard. The kit has a detection sensitivity limit of
1.56 µM lysine.

Assay Principle
Cell Biolabs’ Lysine Assay Kit measures lysine within food or biological samples. Lysine is converted
by lysine oxidase into 6-amino-2-oxohexanoate plus ammonia and hydrogen peroxide. The hydrogen
peroxide is then detected with a highly specific fluorometric probe. Horseradish peroxidase catalyzesthe reaction between the probe and hydrogen peroxide, which bind in a 1:1 ratio. Samples andstandards are read with a standard 96-well fluorometric plate reader. Samples are compared to a known
concentration of lysine standard within the 96-well microtiter plate format (Figure 1).

Figure 1. Lysine Assay Principle.

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

MET-5053: Total Thiol Assay Kit
MET-5055: L-Amino Acid Assay Kit (Fluorometric)
MET-5071: Taurine Assay Kit
MET-5070: Glycine Assay Kit
STA-674: Glutamate Assay Kit (Fluorometric)

Kit Components
1.
2.
3.
4.
5.

Lysine Standard (Part No. 51291C): One 50 µL tube at 10 mM.
10X Assay Buffer (Part No. 51192A): One 30 mL bottle.
Fluorometric Probe (Part No. 50231C): One 50 μL amber tube.
HRP (Part No. 234402): One 100 μL tube at 100 U/mL in glycerol.
Lysine Oxidase (Part No. 51292D): One 50 µL tube containing recombinant Lysine Oxidase fromTrichoderma viride.

Materials Not Supplied
1. Distilled or deionized water
2. 1X PBS
3. Standard 96-well black microtiter plate and/or cell culture microplate

StorageUpon receipt, store the 10X Assay Buffer at room temperature. Store the Lysine Oxidase at -80ºC.
Store all other components at -20ºC. The Fluorometric Probe is light sensitive and must be storedaccordingly. Avoid multiple freeze/thaw cycles.

Preparation of Reagents••

1X Assay Buffer: Dilute the 10X Assay Buffer to 1X with deionized water. Mix to homogeneity.
Store the 1X Assay Buffer at 4ºC.
Reaction Mix: Prepare a Reaction Mix by diluting the Fluorometric Probe 1:100, HRP 1:500, andLysine Oxidase 1:100 in 1X Assay Buffer. For example, add 10 μL Fluorometric Probe stocksolution, 2 μL HRP stock solution, and 10 µL of Lysine Oxidase to 978 μL of 1X Assay Buffer fora total of 1 mL. This Reaction Mix volume is enough for 20 assays. The Reaction Mix is stable for1 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.
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.
3••Saliva or Urine: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. Dilute thesupernatant 1:10 to 1:20 in 1X Assay Buffer just prior to performing the assay.
Serum or Plasma: Deproteinate the sample by running it through a centrifugal filter unit (e.g.
Amicon Ultra 0.5 mL 10K Cat. No. UFC501024) and collecting the flow through. To remove
insoluble particles, centrifuge at 10,000 rpm for 5 min. Dilute the supernatant as necessary into 1X
Assay Buffer just prior to performing the assay.

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 willoxidize the Fluorometric Probe and could result in erroneous readings. To minimize this interference, itis 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 stable in thepresence of thiols (above 10 μM).

Preparation of Standard Curve
Prepare fresh Lysine Standards before use by diluting in 1X Assay Buffer according to Table 2.
Standard Tubes 10 mM Lysine Solution (µL) 1X Assay Buffer (µL)
Lysine (µM)
15495
100
2250 of Tube #125050
3250 of Tube #225025
4250 of Tube #3250
12.5
5250 of Tube #4250
6.25
6250 of Tube #5250
3.13
7250 of Tube #6250
1.56
80250
0Table 2. Preparation of Lysine 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 Lysine 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 10 to
30 minutes at room temperature 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 lysine within samples by comparing the sample fluorescence to thestandard curve.

4Example of Results
The following figures demonstrate typical Lysine Assay Kit results. One should use the data below forreference only. This data should not be used to interpret or calculate actual sample results.

Figure 2: Lysine Standard Curve.

Figure 3: Lysine Detection in human serum, human plasma, or BSA using the Lysine Assay Kit. 50 µL ofneat human serum, human plasma, or 10 mg/mL BSA was tested according to the Assay Protocol.

References
1. Betts MJ, Russell RB (2003). Bioinformatics for Geneticists. pp. 289–316.
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3.
4.
5.
6.
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8.

Votyakova TV, and Reynolds IJ (2001) Neurochem. 79:266.
Kumar S, Tsai CJ, Nussinov R (2000). Prot. Engineer. 13: 179–191.
Sokalingam S, Raghunathan G, Soundrarajan N, Lee SG (2012). PLOS One. 7: e40410
Dambacher S, Hahn M, Schotta G (2010). Heredity. 105: 24–37.
Martin C, Zhang Y (N2005). Nature Rev. Mol. Cell Bio. 6: 838–849.
Shoulders MD, Raines RT (2009). An.Rev. of Biochem. 78: 929–958.
Civitelli R, Villareal DT, Agnusdei D, Nardi P, Avioli LV, Gennari C (1992). Nutrition. 8: 400–405.
9. Flanagan JL, Simmons PA, Vehige J, Willcox MD, Garrett Q (2010). Nutrition & Metabolism. 7:30.

Warranty
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WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CELL BIOLABS’s sole obligation and purchaser’sexclusive 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.

Contact Information
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San Diego, CA 92111
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E-mail: tech@cellbiolabs.com
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