Supplemental Protocols

Deproteination by 50% 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 (TX-100).

(a) tissue:

1. Homogenize 50 mg tissue in 0.2 ml ice-cold dH2O or 0.1% TX-100 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 ~14,000 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 shake tube to mix.

4. Keep solution on ice for 3 min.

5. Centrifuge solution in a microfuge at ~14,000 rpm at 4ºC for 3 min.

6. Harvest supernatant and store at -20ºC.

(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 shake tube to mix. Keep solution on ice for 3 min.

3. Centrifuge solution in a microfuge at ~14,000 rpm at 4°C for 3 min.

4. Harvest supernatant and store at -20°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 -80ºC.

2. Bring up cells in 0.1 ml ice-cold dH2O or 0.1% TX-100. 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 shake tube to mix. Keep solution on ice for 3 min.

5. Centrifuge solution in a microfuge at ~14,000 rpm at 4ºC for 3 min.

6. Harvest supernatant and store at -20ºC.