Supplemental Protocols

Deproteination by PEG: The protocol is based on precipitation of proteins by 50% polyethylene glycol (PEG)-8000. The PEG solution is highly viscous, and should be pipetted slowly using a cut tip. Note that the method is not recommended for analysis of nucleotides. Cells/tissues may be homogenized in ice-cold dH2O or dH2O supplemented with 0.1% Triton X-100.

(a) tissue:

1. Homogenize 50 mg tissue in 0.2 ml ice-cold dH2O on ice. Use the same tissue:dH2O ratio for all samples. A mechanical homogenizer is recommended for this step.

2. Centrifuge solution in a microfuge at ~13,000 rpm at 4ºC for 5 min. Transfer supernatant to a 1.5-ml microtube placed on ice.

3. Add an equal volume of PEG (pipette PEG solution slowly using a cut tip). Vigorously vortex solution to mix for at least 30 sec.

4. Keep solution on ice for 30 min.

5. Centrifuge solution in a microfuge at ~13,000 rpm at 4ºC for 5 min.

6. Harvest supernatant and store at -70ºC.

(b) serum/plasma/urine:

1. Mix 50 ul of each sample with 50 ul PEG Solution in a 1.5-ml microtube (pipette PEG solution slowly using a cut tip).

2. Vigorously vortex solution to mix for at least 30 sec. Keep solution on ice for 30 min.

3. Centrifuge solution in a microfuge at ~13,000 rpm at 4°C for 5 min.

4. Harvest supernatant and store at -70°C. Note that the sample has been diluted 2-fold.

(c) animal cells:

1. Pellet several million cells in a microtube and discard medium. Freeze cell pellet at -70ºC.

2. Bring up cells in 0.1 ml ice-cold dH2O. Use a mechanical or Dounce homogenizer to homogenize cells on ice.

3. Mix homogenate with an equal volume of PEG (pipette PEG solution slowly using a cut tip) in a 1.5-ml microtube.

4. Vigorously vortex solution to mix for at least 30 sec. Keep solution on ice for 30 min.

5. Centrifuge solution in a microfuge at ~13,000 rpm at 4ºC for 5 min.

6. Harvest supernatant and store at -70ºC.