Invitrosol digestion protocol

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Protocol for Trypsin Digest using 80%ACN and Invitrosol

1.Add some buffer (i.e.100mM Tris-HCl, pH 8) to the sample so the sample volume is 100ul.
2.Add 400ul of MeOH and 100ul of Chloroform to the sample.  
3.Vortex and spin at full speed for 5 minutes.
4.Discard the supernatant carefully so you don’t disturb the pellet.  
5.Add 300ul of MeOH to the pellet and repeat step 3-4.
6.Discard as much supernatant as possible without disturbing the protein pellet.
7.Add 5ul of 5X invitrosol to the pellet and gently resuspend the pellet.
8.Heat the sample at 60oC for 5 minutes.
9.Cool the sample down to room temperature and add 45ul of 90% acetonitrile to the sample (so the final concentration of  
  acentonitrile is ~ 80%).
10.Resuspend gently and sonicate for 2 hours in the water bath.
11.Cool down the sample to room temperature and add trypsin (1:50).
12.Place the sample in the 37oC shaker and digest over night.
13.Next day, add 10ul of 90% Formic acid and speedvac to almost dry.  
14.Resuspend the peptides in desired volume of buffer A and load on the column.