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Trypsin Digestion

  •   Stock ammonium bicarbonate buffer, pH 8.0
  •   Lyophilized trypsin
  •   Acetonitrile

  1. Cut the gel sample into 1mm size pieces or smaller. Typically, the smaller the gel segment the better. Place the gel pieces into separate 0.5 ml polypropylene tubes.
  2. Dilute a stock solution ammonium bicarbonate buffer to 50mM. The diluted buffer should be prepared fresh every time. The stock can be prepared and stored indefinitely if kept frozen (< -20 C).
  3. Add 100 ml of ammonium bicarbonate buffer to each tube and incubate the samples at 37° C for 30 min.
  4. After incubation, remove the buffer and add 100 ml of water to each tube. Incubate the samples again at 37° C for 30 min.
  5. After incubation, remove the water and add 100 ml of acetonitrile to each tube to dehydrate the gel pieces. Vortex the samples and after 5 min the acetonitrile is removed. Add 100 ml of acetonitrile again to each of the sample tubes, vortex them, and remove the acetonitrile after 5 min. Be careful not to aspirate the dried gel pieces into the pipette tip as the gel pieces shrink after dehydration and can be pulled into the pipette tip very easily.
  6. To a 20 g vial of lyophilized trypsin add 2ml of 25 mM ammonium bicarbonate, pH 8.0. Mix the solution thoroughly to ensure complete dissolution of the trypsin.
  7. Add the trypsin solution to each sample tube in an volume to just cover the dried gel. The trypsin solution will be pulled into the gel as it swells. If the gel was Coomassie-stained, add more trypsin in order to maintain the level trypsin solution in the tube. The solution should always just cover the top of the gel pieces. If the gel was SYPRO or silver-stained, use ammonium bicarbonate buffer only to maintain the trypsin level.
  8. Incubate the samples at 37° C for exactly 6 hrs. Continuously check the level of the trypsin solution and add additional trypsin solution or buffer as needed.
  9. After digestion, remove the samples from the incubator and spot 1 ml of sample solution directly onto a MALDI target plate. The samples are ready for analysis by MALDI.