Protocols for Protein Precipitation in Samples before Using iTRAQ® Kits

日付: 05/01/2018
カテゴリー: Academia Omics , Pharma CRO , iTRAQ Reagent

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


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 µ.  
  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.