FASP Digestion

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Procedure for FASP Digestion

Before proceeding, review Contamination Issues document.

Solution recipes you will need are in the Digestion Recipes document.

Necessary resources for the digestion protocol are the Digestion Resources document.

⦁ Transfer 150 ug of total protein to a new microcentrifuge tube. 
⦁ Add 200 μL 8 M urea solution and transfer to the filter unit. 
⦁ Centrifuge at 14,000 x g for 20 min. 
⦁ Add an additional 200 μL 8M urea, vortex and centrifuge at 14,000x g for 20 minutes. 
⦁ Discard flow through. 
⦁ Add 100 μL 10 mM DTT solution to filter, vortex and incubate at 37°C for 20 minutes. If your lysis buffer contains DTT, skip this step. 
⦁ Add 100 μL 15 mM IAA solution to filter, vortex and incubate in dark at room temperature for 20 minutes. 
⦁ Centrifuge at 14,000x g for 20 min. 
⦁ Add 100 μL 8M urea, vortex and centrifuge at 14,000x g for 20 minutes. 
⦁ Repeat the previous step. 
⦁ Discard flow through. 
⦁ Add 200 μL of 50 mM AmBic and vortex well. 
⦁ Centrifuge at 14,000x g for 20 min. 
⦁ Transfer the filter unit to a new collection tube. 
⦁ Add 120 μL of Trypsin:Lys-C (1:50 enzyme: protein) mix working solution, gently vortex and incubate for ~16 h at 37°C. 
⦁ After incubation time, transfer the filter units to new collection tubes and centrifuge at 14,000x g for 10 min. 
⦁ Add 50 μL 50 mM AmBic to filter unit and centrifuge at 14,000x g for 20 min. 
⦁ Concentrate the elute in a speed vacuum. PAUSE POINT - Samples can be stored at −80°C for several months in this form).