In-gel Digestion Protocols
In-gel digestion of Coomassie stained bands with reduction and alkylation
- Add 100 uL of 100 mM amm bic. to gel (or enough liquid to cover the gel). Leave for 5-10 minutes.
- 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.
- Repeat steps 1 and 2.
- If gel pieces are still blue, repeat steps 1 and 2
Reduction and Alkylation
- Add 25uL of 50 mM amm bic to gel pieces.
- Add 50 uL of 10mM DTT (made up in 100mM amm bic - make up FRESH).
- 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.
- Remove DTT solution
- Add 25 uL of 50 mM amm bic to gel pieces.
- Add 50 uL of 55mM iodoacetamide (made up in 100mM amm bic - make up FRESH and keep in the dark).
- Incubate at room temperature in the dark for 20 min.
- Remove iodoacetamide solution.
- Wash with 100 uL of 50mM amm bic. Leave for 5 min, then remove.
- Wash with 100 uL of ACN. Leave for 5 min, then remove.
- Repeat this step with occasional vortexing, until gel pieces become opaque white and completely dehydrated, then remove acetonitrile.
- If you have speed vac dry the samples in speedvac for 2-3 minutes to completely evaporate acetonitrile.
- 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.
- Add 75 uL of 50mM amm bic to keep gel pieces hydrated during the digest.
- Incubate overnight at 37°C.
- Remove supernatant - KEEP
- Add 100 uL of 1% formic acid/2% ACN to gel pieces. Leave for 5 min, then transfer to vial containing supernatant.
- Add 100 uL of 1:1 ACN/water. Leave for 5 min, then transfer to vial containing supernatant.
- Add 100 uL of 1% formic acid in ACN. Leave for 5 min, then transfer to vial containing supernatant.
- Dry extracted peptides.
- Desalt extracted peptides using zip-tip or stage-tip.