In-gel Digestion of Excised Protein Spots

(Silver Stained Gels) WITHOUT Destaining



1. Washing/Destaining

  1. Wash gel twice with dd(doubly deionized) H2O for 15 min
  2. Excise bands/spots (cut as close to the band/spot as possible to minimize excess Polyacrylamide gel material), and cut into 1 mm cubes and place them in an eppendorf tube .
  3. Dry samples in a speed vacuum at around 56~60°C for about 45 mins, the gel pieces must shrink and be completely dehydrated.
2. Reduction/Alkylation
  1. Remove samples from the speed vacuum and let cool
  2. Add 40 µl of 10 mM DTT/100 mM Ambic and incubate at 56°C in a water bath or thermocycler for 45 min.
  3. Remove samples and let cool.
  4. Pull off solution and discard, immediately add 40 µl of 55 mM IAA/100 mM Ambic , then incubate at room temperature for 30 mins in the dark.
  5. Pull off solution and wash with 40 µl of 100 mM Ambic , then incubate at room temperature for 5 min.
  6. Add 40 µl of acetonitrile to make 1:1 solution of Ambic and ACN, then incubate at room temperature for 15 min.
  7. Pull off solution and discard, dry gel pieces in speed vacuum as in 1c).

3. Digestion/Extraction

  1. Add 40 µl or enough trypsin solution to cover gel pieces and incubate at 4°C for 45 mins (use ice bath or thermocycler, add more solution if pieces absorb all the liquid).
  2. Pull off excess solution and discard, add 40 µl (or enough to cover gel pieces) same buffer but without trypsin and incubate at 37°C for 16 hrs (overnight).
  3. Pull off supernatant and save at 3°C.
  4. To gel add 20 µl of 25 mM Ambic and incubate at room temperature for 15 min.
  5. Add 20 µl acetonitrile to make 1:1 solution of Ambic/ACN and incubate for 15 min.
  6. Pull off the supernatant and combine it with the one from c).
  7. To gel pieces add 20 µl of 5% Formic Acid , then incubate at room temperature for 15 min.
  8. Add 20 µl 50% ACN to make 1:1 solution of ACN/Formic Acid, then incubate for 15 min.
  9. Repeat 3f), 3g), 3h), 3f).
  10. To pooled supernatant add 10 mM DTT to give final concentration of 1 mM DTT.
  11. Completely dry the supernatant, which is the digested extracts in speed vacuum.
  12. Resuspend the digested extracts in 15 µl~20 µl of 5% Formic Acid for MS or MS/MS analysis.

Note: Larger spots/bands may require more solution. Please adjust the volume accordingly.