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)
- To sample of starting volume 100 µL .
- Add 400 µL methanol
- Vortex well
- Add 100 µL chloroform
- Vortex
- Add 300 µL water
- Vortex
- Spin 1 min at 14,0000 g
- Remove top aqueous layer (protein is between layers)
- Add 400 µL methanol
- Vortex
- Spin 2 min at 14,000 g
- Remove as much MeOH as possible without disturbing the pellet. Pellet will be at the bottom.
- 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:
- Place samples on ice and acetone at -20 ˚C
- Add 6-8 volumes of ice cold acetone to the sample
- Vortex
- Allow to sit and settle for 30-50 min on ice (see flocculent). Alternatively, refrigerate samples at 4 ˚C.
- Spin at 2000 rpm for 5 min
- 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 :
- 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).)
- Precipitate at -20 °C for 1 hr.
- Centrifuge at 18,000 x g for 15 min at 4 °C in a micro centrifuge.
- 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.
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