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Amplite® Choline Quantitation Kit

Choline dose response was obtained with Amplite® Choline Quantitation Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 100 nM (10 picomole/well) of choline can be detected with 30 minutes incubation time (n=3).
Choline dose response was obtained with Amplite® Choline Quantitation Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 100 nM (10 picomole/well) of choline can be detected with 30 minutes incubation time (n=3).
Choline dose response was obtained with Amplite® Choline Quantitation Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 100 nM (10 picomole/well) of choline can be detected with 30 minutes incubation time (n=3).
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Spectral properties
Excitation (nm)571
Emission (nm)584
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Excitation (nm)
Emission (nm)
Choline and its metabolites play an important role in the structural integrity and signaling of cell membranes and cholinergic neurotransmission (acetylcholine synthesis). It is a major source for methyl groups via its metabolite, trimethylglycine that participates in the S-adenosylmethionine synthesis pathways. Choline deficiency may cause liver disease, atherosclerosis and possibly neurological disorders. Despite its importance in the central nervous system as a precursor for acetylcholine and membrane phosphatidylcholine, the role of choline in mental illness has been little studied. This Amplite® Choline Quantitation Kit provides one of the most sensitive methods for quantifying choline. The kit uses Amplite® Red to quantify the concentration of choline, which is related to the production of hydrogen peroxide in the choline oxidase-mediated enzyme coupling reactions. The amount of choline is proportional to the concentration of hydrogen peroxide formed in the enzyme coupling reaction cycle. In the presence of peroxidase, the fluorescence intensity of Amplite® Red is proportional to the formation of hydrogen peroxide that is converted to the concentration of choline. The assay can be readily read with a fluorescence microplate reader. Alternatively the assay can also be read at ~570 nm with an absorption microplate reader.


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol Summary
  1. Prepare and add choline standards and/or test samples (50 µL)
  2. Prepare and add Choline Assay working solution (50 µL)
  3. Incubate at room temperature for 15-60 minutes
  4. Monitor fluroscence intensity at Ex/Em = 540/590 nm
Important Note

Thaw all the kit components at room temperature before starting the experiment.


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Amplite™ Red stock solution (250X)

Add 40 µL of DMSO (Component E) into the vial of Amplite™ Red substrate (Component A). The stock solution should be used promptly. Any remaining solution should be aliquoted and refrozen at -20 oC. Note: Avoid repeated freeze-thaw cycles. Note: The Amplite™ Red substrate is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM. The Amplite™ Red substrate is also unstable at high pH (> 8.5). Therefore, the reaction should be performed at pH 7–8. The provided Assay Buffer (pH 7.4) is recommended.

Choline standard stock solution (50 mM)

Add 400 µL of ddH2O into the vial of Choline Standard (Component C) and mix well. Note: The unused choline stock solution should be divided into single use aliquotes and stored at -20oC.


For convenience, use the Serial Dilution Planner:

Choline standard
Add 20 µL of Choline standard stock solution (50mM) to 980 µL Assay Buffer (Component D) to generate 1000 µM standard solution. Note: Diluted choline standard solution is unstable, and should be used within 4 hours. Take 30 µL of 1000 µM standard to 970 µL Assay Buffer (Component D) to generate 30 µM choline standard solution, and then perform 1:3 serial dilutions to get 10, 3, 1, 0.3, 0.1, 0.03, and 0 µM choline standard. (Refer link in the Preparation of Standard Solution)


Choline Assay working solution

Add 5 mL of Assay Buffer (Component D) into the bottle of Choline Probe (Component B), and mix them well. Add 20 µL of Amplite Red™ stock solution (250X) into the Choline Probe.


Table 1. Layout of Choline standards and test samples in a solid black 96-well microplate. CS = Choline standard (CS1-CS7); BL = blank control; TS = test sample.


Table 2. Reagent composition for each well

CS1 - CS750 µLSerial Dilutions (0.03 to 30 µM)
BL50 µLAssay Buffer (Component D)
TS50 µLtest sample
Choline assay
  1. Add choline standards and choline containing test samples into a 96-well solid black microplate as described in Tables 1 and 2.
  2. Add 50 µL of Choline Assay working solution into each well of choline standard, blank control, and test samples (Table 2) to make the total choline assay volume of 100 µL/well. Note: For a 384-well plate, add 25 µL of sample and 25 µL of assay reaction mixture into each well.
  3. Incubate the reaction for 15 to 60 minutes at room temperature, protected from light.
  4. Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em= 530-570 nm/590-600 nm (optimal Ex/Em = 540/590 nm).


Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)571
Emission (nm)584



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Journal: Scientific reports (2021): 1--16
Structural snapshots of the catalytic cycle of the phosphodiesterase Autotaxin
Authors: Hausmann, Jens and Keune, Willem-Jan and Ederveen, Agnes L Hipgrave and van Zeijl, Leonie and Joosten, Robbie P and Perrakis, Anastassis
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View all 62 references: Citation Explorer
Real-time multiplex PCR assay for genotyping of three apolipoprotein E alleles and two choline acetyltransferase alleles with three hybridization probes
Authors: Park HD, Park KU, Kim KW, Song J, Chang HE, Heo SR, Lee HJ, Kim JQ.
Journal: Clin Chem Lab Med (2007): 346
[Methyl-3H]-choline incorporation into MCF-7 cells: correlation with proliferation, choline kinase and phospholipase D assay
Authors: Al-Saeedi F, Smith T, Welch A.
Journal: Anticancer Res (2007): 901
Rapid high-throughput detection of peroxide with an acridinium-9-carboxamide: a homogeneous chemiluminescent assay for plasma choline
Authors: Adamczyk M, Brashear RJ, Mattingly PG.
Journal: Bioorg Med Chem Lett (2006): 2407
A simple and rapid radiochemical choline acetyltransferase (ChAT) assay screening test
Authors: Shiba K, Ogawa K, Kinuya S, Yajima K, Mori H.
Journal: J Neurosci Methods (2006): 98
Comparison of different sperm detection methods: amplification of fragment 3 of amelogenin gene (AMY 3), acid phosphatase detection, microcristalline choline assay and microscopic examination
Authors: Rydzewska MJ, Janica J, Pepinski W, Skawronska M, Niemcunowicz-Janica A.
Journal: Rocz Akad Med Bialymst (2000): 63
Rapid assay of choline in foods using microwave hydrolysis and a choline biosensor
Authors: Panfili G, Manzi P, Compagnone D, Scarciglia L, Palleschi G.
Journal: J Agric Food Chem (2000): 3403
Establishment of an efficient enzyme-linked immunosorbent assay for the determination of human choline acetyltransferase
Authors: Poethke R, Tumani H, Felgenhauer K, Mader M.
Journal: J Neuroimmunol (1997): 206
Ion-exchange liquid chromatography method with indirect UV detection for the assay of choline in pharmaceutical preparations
Authors: Leroy P, Barbaras M, Colin JL, Nicolas A.
Journal: J Pharm Biomed Anal (1995): 581
Assay, Purification, and Partial Characterization of Choline Monooxygenase from Spinach
Authors: Burnet M, Lafontaine PJ, Hanson AD.
Journal: Plant Physiol (1995): 581
The identification of human semen by a chemiluminescent assay of choline
Authors: Manabe F, Tsutsumi A, Yamamoto Y, Hashimoto Y, Ishizu H.
Journal: Nihon Hoigaku Zasshi (1991): 205