BioProtocols--Met3

CHOLINE INCORPORATION ASSAY

Materials Needed:

Model Specifications:

Procedures:

Obtain the samples from the 4oC refrigerator.
Place the samples on ice and note the amount of distilled water added per tube.
Sonicate the samples for intervals of 20 seconds or less, with waiting periods on ice until the sample has been completely mixed

Note: Notice the appearance of the tissue to ensure all tissue has been sonicated. If cells are used,

sonicate once or twice to ensure proper sonication since the cells cannot be seen with the

naked eye.

Transfer the samples to glass tubes
1. Save 50uL of the solution into 12x75mm glass tubes for DNA Assay (follows a different protocol)

OR

Save 50uL of the solution into 12x75mm glass tubes for the Bradford Protein Assay (follows a different protocol)

Transfer the remaining 950uL of the solution in 13x100mm glass tubes for lipid extraction

Add 2mL of chloroform and 1mL of methanol to each tube.
Vortex for approximately 5 seconds.
Refrigerate for 15min to 1 hr.
Carefully transfer the bottom layer to new, properly labeled, glass tubes.
Add 2mL of chloroform to the original tubes
Vortex
Refrigerate for 15min to 1hr.
Transfer the bottom layer to the new tubes with the previously collected lipid portion.
Dry the samples under N₂ at 60oC.
Add 0.5mL Osmuim
Wash the sides of the tube with osmium during a 15 min wait