IN-GEL TRYPTIC DIGESTION
IN A 96-WELL MICROTITER PLATE
FOR COOMASSIE BLUE STAINED GELS

96-Well Microplate:   Nunc

V-96 Polypropylene Plate Purchased from Fisher Scientific. Cat No. 12-565-263

Special Pipetter: Multichannel Pipetter

Solutions:

  • 0.1% TFA/60% CH3CN

  • 50% H2O/ 50% CH3CN

  • 1 M NH4HCO3 (stock solution)

  • 50% CH3CN / 50 mM NH4HCO3

  • 100 mM NEM acetate pH 8.6 (N-Ethyl Morpholine, Aldrich stock = 7.86 M prepare fresh; adjust pH to 8.6 with 1M acetic acid)

  • 50% CH3CN / 30 mM NEM acetate pH 8.6

  • 30 mM NEM acetate pH 8.6

  • Trypsin (modified, Promega)

Procedure

  1. Prewash 96-well plate with 3 X 150 µl of 0.1% TFA/60% acetonitrile.

  2. Cut the sample gel pieces into ~1 mm3 cubes and put them into each well on the prewashed 96-well plate separately. Do not use too much gel. Limit gel to ~9 mm3.

  3. Add 2X 250 µl of 50%H2O/ 50% acetonitrile and wash for 30 minutes.

  4. Remove wash.

  5. Wash gel pieces with 150 µl of 50% acetonitrile/ 50 mM NH4HCO3 for 30 minutes at room temperature on tilt table.

  6. Remove wash.

  7. Wash gel pieces with 3 X 150 µl of 50% acetonitrile / 30 mM NEM acetate pH 8.6 for 30 minutes at room temperature on tilt table.

  8. Remove wash.

  9. Cover the plate with parafilm, and ventilate by puncturing the film over each well, then Speedvac gel pieces to dry completely.

  10. a) Add 0.1 mg modified trypsin (Promega) per 15 mm3 of gel in 15 µl of 30 mM NEM acetate pH 8.6.

    b) Let stand for 5-10 minutes to allow enzyme/buffer to absorb into gel.

  11. Add an additional 10-15 µl of 15 mM NEM acetate pH 8.6 that does not contain enzyme to just cover the gel pieces.

  12. Seal the plate well and incubate overnight at 37°C.

  13. Extract the peptides 2x by adding 50 µl of 0.1% TFA/60% acetonitrile and shaking at room temperature for 30 minutes.

  14. Transfer both extracts to a well in another prewashed 96-well plate.

  15. Speedvac the extracts to dryness.

  16. Reconstituted in 0.1% TFA / 10% acetonitrile for subsequent MS analysis.