In Gel Digestion Protocols for Silver Stained and Coomassie Stained Gels

Silver stain in-gel digestion (EMBL protocol)

1. Washing

  1. Before excising bands wash gels twice in ddH2O for 15 minutes.
  2. Excise bands, cut as close to the band as possible to minimize excess gel material and cut into 1 mm cubes, place in Eppendorf tube.
  3. Dry samples in speed vac for approximately 15 minutes (must be very dry).

2. Alkylation

(DO NOT need to do, if only obtaining protein identification)

  1. Remove samples from speed vac and let cool.
  2. Add 40 µL of 10 mM DTT/100 mM Ambic and incubate in water bath at 56ˇăC for 45 minutes.
  3. Remove samples from bath and let cool.
  4. Pull off solution and immediately add 40 µL 55 mM iodoacetamide/100 mM Ambic and incubate at room temperature for 30 minutes in the dark.
  5. Pull off solution and wash with 40 µL of 100mM Ambic incubate 5 minutes.
  6. Add 40 µL of ACN to make 1:1 soln and incubate for 15 minutes.
  7. Pull off solution and dry gel pieces in speed vac for 15 minutes

3. Digestion

  1. Remove samples from speed vac and let cool.
  2. Add 40 µL (enough to cover pieces) of Trypsin solution and incubate 45 min at 4ˇăC (ice bath). Add more solution if pieces absorb all of the liquid.
  3. Pull off excess solution and discard, add 10 µL of same buffer without trypsin (enough to cover gel pieces) and incubate overnight at 37ˇăC.
  4. Pull supernate and save, to gel add 20 µL of 25 mM Ambic and incubate 15 minutes.
  5. Add same amount of ACN to make 1:1 soln of Ambic/ACN and incubate 15 minutes.
  6. Pull off supernate and add to solution saved in 3(c), to gel add 20 µL of 5% formic acid and incubate 15 minutes.
  7. Add same amount of ACN and incubate 15 minutes.
  8. Pull off supernate and pool with 3(c) soln, to gel add 20 µL of 5% formic acid and incubate for 15 minutes.
  9. Add same amount of ACN and incubate 15 minutes.
  10. Pull off supernate and pool with 3(c) solution.
  11. To pooled solution in 3(c) add 10 mM DTT to give final concentration of 1 mM DTT.
  12. Vacuum dry solution in 3(c) almost to dryness.
  13. Resuspend in 15 µL of 5% formic acid for mass spectrometric analysis.

NOTES:

  • Incubations are at room temperature unless otherwise noted.
  • Larger bands will require more solution volume.

Coomassie in-gel digestion (EMBL protocol)

1. Washing

  1. Before excising bands, wash gels twice in ddH2O for 15 minutes.
  2. Excise bands, cut as close to the band as possible to minimize excess gel material, cut into 1 mm cubes, and place in Eppendorf tube.
  3. To band add 100 µL of ddH2O and incubate for 15 min.
  4. Pull off ddH2O and add 40 µL of 50/50 acetonitrile (ACN)/ddH2O and incubate 15 minutes.
  5. Pull off solution and add 40 µL of acetonitrile, incubate until gel pieces are white and sticky.
  6. Pull off solution and add 40 µL of 100 mM ammonium bicarbonate (ambic), incubate 5 minutes.
  7. Add 40 µL of ACN to make 1:1 solution, incubate 15 minutes.
  8. Pull off soln and dry samples in speed vac for approximately 15 minutes (must be very dry).

2. Alkylation

(DO NOT need to do, if only obtaining protein identification)

  1. Remove samples from speed vac and let cool.
  2. Add 40 µL of 10 mM DTT/100 mM Ambic and incubate in water bath at 56ˇăC for 45 minutes.
  3. Remove samples from bath and let cool.
  4. Pull off solution and immediately add 40 µL of 55 mM iodoacetamide/100 mM Ambic and incubate at room temperature for 30 minutes in the dark.
  5. Pull off solution and wash with 40 µL of 100 mM Ambic incubate 5 minutes.
  6. Add 40 µL of ACN to make 1:1 soln and incubate for 15 minutes.
  7. Pull off solution and dry gel pieces in speed vac for 15 minutes

3. Digestion

  1. Add 40 µL (enough to cover pieces) of Trypsin solution and incubate 45 min at 4ˇăC (ice bath). Add more solution if pieces absorb all of the liquid.
  2. Pull off excess solution and discard, add 10µL of same buffer without trypsin (enough to cover gel pieces) and incubate overnight at 37ˇăC.
  3. Pull supernate and save, to gel add 20 µL of 25 mM Ambic and incubate 15 minutes.
  4. Add same amount of ACN to make 1:1 soln of Ambic/ACN and incubate 15 minutes.
  5. Pull off supernate and add to solution saved in 3(c), to gel add 20 µL of 5% formic acid and incubate 15 minutes.
  6. Add same amount of ACN and incubate 15 minutes.
  7. Pull off supernate and pool with 3(c) soln; to gel add 20 µL of 5% formic acid and incubate for 15 minutes.
  8. Add same amount of ACN and incubate 15 minutes.
  9. Pull off supernate and pool with 3(c) solution.
  10. To pooled solution in 3(c) add 10 mM DTT to give final concentration of 1 mM DTT.
  11. Completely dry 3(c) solution in speed vac.
  12. Resuspend in 15 µL of 5% formic acid for mass spectrometric analysis.

NOTES:

  • Incubations are at room temperature unless otherwise noted.
  • Larger bands will require more solution volume.

    Solutions

    50% ACN in ddH2O
    250 µL of ACN + 250 µL of ddH2O
    100 mM ammonium bicarbonate (Ambic)
    100 µL of 1M Ambic in 900 µL of ddH2O
    10 mM DTT in 100 mM Ambic
    5 µL of 2 M DTT + 895 µL of ddH2O + 100 µL of 1 M Ambic
    55 mM IAA in 100 mM Ambic
    10 mg of iodoacetamide + 100 µL of 1 M Ambic + 900 µL of ddH2O
    50 mM Ambic in 5 mM CaCl2 with 12.5 ng of Trypsin
    5 µL of 1 M CaCl2 + 50 µL of 1 M Ambic + 945 µL of ddH2O
    Take100 µL of above solution add 12.5 µL of 0.1 mg/µL Trypsin solution
    25 mM Ambic
    25 µL of 1 M Ambic + 975 µL of ddH2O
    10 mM of DTT in ddH2O
    5 µL of 2 M DTT + 995 µL of ddH2O
    5% formic acid
    57 µL of stock formic acid (88%) + 943 µL of ddH2O