1. Digest protein.
   1. Prepare 20 ug/mL trypsin in 20 mM ammonium bicarbonate.  Use only sequencing grade trypsin, which can be purchased from Sigma or Promega.  We purchase Trypsin Gold from Promega (cat. no. V5280).
      
   1. Prepare a 1 mg/mL stock of trypsin in acetic acid buffer.  For Promega Trypsin Gold, resuspend 100 ug lyophilized trypsin in 100 uL acetic acid buffer.
      
   2. Aliquot 10 uL to 10 microfuge tubes and store at -80^oC.
      
   3. Dilute each 10 uL aliquot with 500 uL of 20 mM ammonium bicarbonate.
      

      15.8 mg ammonium bicarbonate
      10 mL H₂O
      

   2. Add 10 uL trypsin solution to each gel plug (for 2.0 mm diameter gel plugs).  Seal plate/tube.  If working with gel bands, increase volume relative to gel amount.
      
      
   3. Incubate gel plugs at 37^oC for 2 hours-overnight.
      
      
2. Extract and dry peptides.
   1. Add 60 uL 50% acetonitrile/0.1% TFA to each gel plug (for 2.0 mm diameter gel plugs).  Incubate at room temperature for 20 minutes.  If working with gel bands, increase volume relative to gel amount.
      

500 uL acetonitrile
500 u L H₂O
1 uL TFA

2. Remove extract solution and save.
   
   
3. Add 40 uL 50% acetonitrile/0.1% TFA to each gel plug (for 2.0 mm diameter gel plugs).  Incubate at room temperature for 20 minutes.  If working with gel bands, increase volume relative to gel amount.
   
   
4. Remove extract solution and combine with earlier saved extract solution.
   
   
5. Dry combined extract solution.  Sample can be shipped overnight at ambient temperature.

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