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
