Amplite® Choline Quantitation Kit
Price | |
Catalog Number | |
Unit Size | |
Quantity |
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
Quotation | Request |
International | See distributors |
Shipping | Standard overnight for United States, inquire for international |
Excitation (nm) | 571 |
Emission (nm) | 584 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | ![]() ![]() |
Excitation (nm) 571 | Emission (nm) 584 |
Platform
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Cutoff | 570 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare and add choline standards and/or test samples (50 µL)
- Prepare and add Choline Assay working solution (50 µL)
- Incubate at room temperature for 15-60 minutes
- Monitor fluroscence intensity at Ex/Em = 540/590 nm
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. 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.
2. 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.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/40007
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)
PREPARATION OF WORKING 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.
SAMPLE EXPERIMENTAL PROTOCOL
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.
BL | BL | TS | TS |
CS1 | CS1 | ... | ... |
CS2 | CS2 | ... | ... |
CS3 | CS3 | ||
CS4 | CS4 | ||
CS5 | CS5 | ||
CS6 | CS6 | ||
CS7 | CS7 |
Table 2. Reagent composition for each well
Choline Standard | Blank Control | Test Sample |
Serial Dilutions: 50 µL | Assay Buffer (Compound B): 50 µL | 50 µL |
Choline assay
- Add choline standards and choline containing test samples into a 96-well solid black microplate as described in Tables 1 and 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.
- Incubate the reaction for 10 to 30 minutes at 37 oC, protected from light.
- Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em= 530-570 nm/590-600 nm (optimal Ex/Em = 540/590 nm).
Product Family
Name | Excitation (nm) | Emission (nm) |
Amplite® Ethanol Quantitation Kit | 571 | 584 |
Amplite® Cholesterol Quantitation Kit | 571 | 584 |
Images
Citations
Authors: Shida, Yukari and Endo, Hitoshi and Owada, Satoshi and Inagaki, Yutaka and Sumiyoshi, Hideaki and Kamiya, Akihide and Eto, Tomoo and Tatemichi, Masayuki
Journal: Scientific reports (2021): 1--16
Authors: Gold, Sarah Ruth
Journal: (2020)
Authors: Hausmann, Jens and Keune, Willem-Jan and Ederveen, Agnes L Hipgrave and van Zeijl, Leonie and Joosten, Robbie P and Perrakis, Anastassis
Journal: Journal of structural biology (2016)
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