This protocol was developed for 2D gel spots, ~100 ng of protein, using a lower concentration. of trypsin for silver, sypro ruby or faint Coomassie stained bands. If Coomassie stained spots at μg levels (medium to dark) are being digested, use a higher concentration of trypsin. Ideally, samples should be run on 1.0mm acrylamide mini gels. Gradient gels can be helpful for sharpening bands. Cut out the gel piece close to the sides of the band. The goal is to get as much protein in as small an area as possible.

Minimize keratin contamination through the use of proper lab equipment and, if available, a laminar flow hood. Do not use gel trays for staining/destaining that have been previously used for Western blots due to the likelihood of BSA or casein contamination.

Solutions Required

Prepare DTT, iodoacetamide, and trypsin solutions fresh before use. Volumes may vary depending on the amount of gel present.

  • Destain solution (400 uL/sample): 50% methanol, 5% acetic acid in HPLC water
  • Trypsin solution (see below) Trypsin can be purchased at the ICMB Supply Center's ThermoFisher automated freezer.
  • Acetonitrile (600 uL/sample)
  • 100mM ammonium bicarbonate (200 uL/sample):  0.79g of NH4HCO3 in 100mL of HPLC Water, syringe filtered
  • 10mM DTT (100 uL/sample): 1.54mg of DTT in 1ml of 100mM NH4HCO3
  • 50 mM iodoacetamide (100uL/sample):  9 mg of iodoacetamide in 1 mL 100 mM NH4HCO3
  • 50 mM ammonium bicarbonate
  • Extraction buffer 1 (20 uL/sample): 5% formic acid in HPLC water
  • Extraction buffer 2 (20 uL/sample): 1:3 (v:v) 5% Formic Acid in HPLC water : acetonitrile



Trypsin preparation

Depending on the amount of protein in the sample, use the appropriate concentration of trypsin:

Regular strength (final concentration 10 ng/uL)

Promega : Add 160 uL of Promega trypsin resuspension buffer to trypsin stock solution tube (stock: 20 ug in 40 uL, 0.5 ug/uL). Make 20 uL aliquots (2 ug; 0.1 ug/ul of trypsin) and store in the freezer.

Pierce(Thermo Fisher ): Add 200 uL of 50mM acetic acid to a vial of lyophilized trypsin (20 ug/vial). Make 20uL aliquots (2 ug; 0.1 ug/ul of trypsin)   and store in the freezer.

Double strength (final concentration 20 ng/uL)

Promega: Add 60 uL of Promega  trypsin resuspension buffer to trypsin stock solution tube (stock: 20 ug in 40 uL, 0.5 ug/uL). Make 20 uL aliquots (4 ug; 0.2 ug/ul of trypsin) and store in the freezer.

Pierce(Thermo Fisher ): Add 100 uL of 50mM acetic acid to a vial of lyophilized trypsin (20 ug/vial). Make 20 uL aliquots (4 ug; 0.2 ug/ul of trypsin)   and store in the freezer.

Before use, add 180 uL of 50 mM ammonium bicarbonate to the appropriate 20 uL aliquot. (Final concentration: 10 ng/uL or 20 ng/uL) 

Sample digestion

Day 1

  1. Chop the gel band with a clean razor blade into square pieces, 1-2 mm cubes. (Do not macerate the bands)
  2. Place gel pieces into Eppendorf tube.
  3. Cover the gel pieces with destain solution and leave overnight. Spots can be left in destain solution indefinitely at 4oC for long term storage.

Day 2 

Spin down tubes before each addition.

  1. Remove and discard destain solution.
  2. Cover gel pieces with 100% acetonitrile to dehydrate. Remove and repeat to dehydrate gel. Gel pieces should appear white.
  3. Remove and discard 100% acetonitrile solution.
  4. Air dry at room temperature for ~15 minutes.
  5. Cover gel pieces with 10 mM DTT solution to reduce the gel. Spin down or tap tube to remove air bubbles from between pieces.
  6. Incubate for 30 minutes at room temperature.
  7. Remove and discard DTT solution.
  8. Cover gel pieces with 50 mM iodoacetamide solution to alkylate the gel. Spin down or tap tube to remove air bubbles from between pieces.
  9. Incubate for 30 minutes at room temperature. Place into a dark area to minimize exposure to light.
  10. Remove and discard iodoacetamide solution.
  11. Cover gel pieces with 100 mM ammonium bicarbonate solution. Vortex and spin down.
  12. Incubate for 10 minutes at room temperature.
  13. Remove and discard ammonium bicarbonate solution. Repeat ammonium bicarbonate wash one time.
  14. Wash with 100% acetonitrile to dehydrate the gel. Incubatee for 5 minutes at room temperature. Remove and repeat. Gel pieces should appear white
  15. Air dry at room temperature for ~15 minutes..
  16. Prepare trypsin solution (See solution preparation for recipe). Choose either regular or double strength based on amount of protein in gel band.
  17. Add trypsin solution to cover the gel pieces. Tap tube to remove air bubbles from between the pieces. Keep on ice until the gel is fully rehydrated (clear, no longer white).  If gel pieces are not fully hydrated after 10-15 minutes, add a small amount of the trypsin solution.
  18. Add 50 mM ammonium bicarbonate solution to each gel sample so that the gel is fully covered in solution.
  19. Incubate overnight at 37°C.

Day 3 

Spin down tubes before each addition.

  1. Cover the gel pieces with extraction buffer 1, vortex, and spin down.
  2. Cover with parafilm or lid clip and sonicate for 10-15 min.
  3. Transfer liquid to a clean Eppendorf tube.
  4. Cover the gel pieces with extraction buffer 2, vortex, and spin down.
  5. Transfer liquid to the same tube as in step 2.

Samples are now ready for ZipTip protocol.

  • No labels

Confluence Documentation | Web Privacy Policy | Web Accessibility