CHOLINE INCORPORATION ASSAY
Materials Needed:
- Chloroform Chromasolv, HPLC Grade 每 Cat # 366927-1L [Sigma]
- Methanol, HPLC
Grade 每 Cat # A452-4 [Fisher]
- DL-汐-Phosphatidylcholine Dipalmitoyl 每 Cat # P-6769 [Sigma]
- Disposable Culture Tubes (13x100mm) 每
Cat # 14-961-27 [Fisher]
- Disposable Culture Tubes (12x75mm) 每 Cat # 14-961-26 [Fisher]
- Parafilm 每 Cat # 13-374-12 [Fisher]
- Industrial
Grade Nitrogen (CGA 580: Size 200) 每 Cat # NIT3 [Westair]
- Wheaton HDPE Sampule 每 Cat # 03-341-25A [Fisher]
Model Specifications:
- Misonix
Microson Ultrasonic Cell Disruptor
- Organomation N-EVAP Nitrogen Evaporator
- Fisher Vortex Genie 2
- Beckman Coulter LS6500 Multipurpose
Scintillation Counter
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
- 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