For research use only. Not for use in diagnostic procedures.

Answer

To prepare samples prior to iTRAQ® reagent labeling, use the following protocols

1)  Protocol for chloroform/methanol precipitation (for removal of salt, detergent and lipids)

1. To sample of starting volume 100 µL  .
2. Add 400 µL methanol
3. Vortex well
4. Add 100 µL chloroform
5. Vortex
6. Add 300 µL water
7. Vortex
8. Spin 1 min at 14,0000 g
9. Remove top aqueous layer (protein is between layers)
10. Add 400 µL methanol
11. Vortex
12. Spin 2 min at 14,000 g
13. Remove as much MeOH as possible without disturbing the pellet. Pellet will be at the bottom.
14. Use the Speed-Vac to bring the sample to dryness. Alternatively,  let stand in the hood or use a dry fan at only 5 psi. The pellet does not need to be dried completely, and this way it is easier to get the proteins back in solution.

2)  Acetone precipitation for proteins:

1. Place samples on ice and acetone at -20 ˚C
2. Add 6-8 volumes of ice cold acetone to the sample
3. Vortex
4. Allow to sit and settle for 30-50 min on ice (see flocculent). Alternatively, refrigerate samples at 4 ˚C.
5. Spin at 2000 rpm for 5 min
6. Remove supernatant and re-suspend pellet

• Note: This protocol works well for urea containing samples.
• GuHCL containing samples need be diluted. GuHCL to < 2 M with distilled water.
• Re-suspend the acetone pellet in 1 M TEAB (Triethylammonium bicarbonate).

3)  Acetone/trichloroacetic acid (TCA) precipitation :

1. Prepare cell lysate/ice-cold acetone/TCA in a 1:8:1 ratio and invert tube after adding each component. (As an example, add 120 µL cell lysate/protein to 960 µL100% ice-cold acetone and add 120 µL 100% trichloroacetic acid (100% TCA, w/v).)
2. Precipitate at -20 °C for 1 hr.
3. Centrifuge at 18,000 x g for 15 min at 4 °C in a micro centrifuge.
4. Discard supernatant. Wash with 300 µL ice-cold acetone and then re-suspend pellet completely. Centrifuge at 18,000 x g for 15 min at 4 °C.