Proteome Exploration Laboratory
In-gel Digestion Protocols

In-gel digestion of Coomassie stained bands with reduction and alkylation

De-staining

  1. Add 100 uL of 100 mM amm bic. to gel (or enough liquid to cover the gel). Leave for 5-10 minutes.
  2. Remove buffer with pipette and add 50 uL of 1:1(v/v) 50mM amm bic/ACN to gel pieces. Leave for 5-10 minutes.
  3. Repeat steps 1 and 2.
  4. If gel pieces are still blue, repeat steps 1 and 2

Reduction and Alkylation

  1. Add 25uL of 50 mM amm bic to gel pieces.
  2. Add 50 uL of 10mM DTT (made up in 100mM amm bic - make up FRESH).
  3. Incubate at 50°C for 30 min. If gel pieces are large, use more volume but keep the ratio of 10mM DTT to amm bic the same.
  4. Remove DTT solution
  5. Add 25 uL of 50 mM amm bic to gel pieces.
  6. Add 50 uL of 55mM iodoacetamide (made up in 100mM amm bic - make up FRESH and keep in the dark).
  7. Incubate at room temperature in the dark for 20 min.
  8. Remove iodoacetamide solution.

Trypsin Digestion

  1. Wash with 100 uL of 50mM amm bic. Leave for 5 min, then remove.
  2. Wash with 100 uL of ACN. Leave for 5 min, then remove.
  3. Repeat this step with occasional vortexing, until gel pieces become opaque white and completely dehydrated, then remove acetonitrile.
  4. If you have speed vac dry the samples in speedvac for 2-3 minutes to completely evaporate acetonitrile.
  5. Add 25 ul of 6ng/ul trypsin to cover all gel pieces. Leave it for 15-20 minutes and observe that majority of trypsin has been absorbed by the gel pieces, remove extra trypsin.
  6. Add 75 uL of 50mM amm bic to keep gel pieces hydrated during the digest.
  7. Incubate overnight at 37°C.

Extraction

  1. Remove supernatant - KEEP
  2. Add 100 uL of 1% formic acid/2% ACN to gel pieces. Leave for 5 min, then transfer to vial containing supernatant.
  3. Add 100 uL of 1:1 ACN/water. Leave for 5 min, then transfer to vial containing supernatant.
  4. Add 100 uL of 1% formic acid in ACN. Leave for 5 min, then transfer to vial containing supernatant.
  5. Dry extracted peptides.
  6. Desalt extracted peptides using zip-tip or stage-tip.