Amplite® Choline Quantitation Kit

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

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 -20^oC.

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

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 10 to 30 minutes at 37 ^oC, 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).