FASP Kit Protocol-ORNL Developed for Bacteriophage
Prepare Urea/SDS Solution.
Transfer Urea solution + sample to spin filter.
Centrifuge at 14,000 x g for 15 min.
Add 200µL fresh Urea solution (no DTT, no SDS).
Centrifuge at 14,000 x g for 15 min.
Discard flow­‐through.
Add 10µL iodoacetamide solution and 90µL Urea solution (no DTT, no SDS).
Vortex for 1 min, then incubate without mixing for 20 min in the dark.
Centrifuge at 14,000 x g for 10 min.
Add 100µL Urea solution (no DTT, no SDS).
Centrifuge at 14,000 x g for 15 min.
Repeat this step twice.
Discard flow­‐through.
Add 100µL 50mM ammonium bicarbonate solution (provided with FASP kit).
Centrifuge at 14,000 x g for 15 min.
Transfer filter to new collection tube.
Add 75µL digestion solution.
Vortex briefly.
Incubate at 37°C for 4 – 18 hours (NO ROCKING). 
Add 40µL 50mM ammonium bicarbonate solution.
Centrifuge at 14,000 x g for 10 min.
Repeat this step once.
Add 50µL 0.5 M sodium chloride solution (provided with FASP kit).
Centrifuge at 14,000 x g for 10 min.
Filtrate contains digested proteins.
Add 170µL* H₂O + formic acid.
Aliquot (150µL x 2)*, freeze
