Procedure for FASP Digestion

Before proceeding, review Contamination Issues

Solution recipes you will need are here.

Necessary resources for the digestion protocol are here.

⦁ 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).