Oct 24, 2025
- Cristina CARDENAL PERALTA1
- 1University of Edinburgh
Protocol Citation: Cristina CARDENAL PERALTA 2025. FASP-IP. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7156kgwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 24, 2024
Last Modified: October 24, 2025
Protocol Integer ID: 102307
Keywords: FASP, CO-IP, Tryptic Digestion, efficient protein digestion, membrane filter, using membrane filter, fasp, mass spectrometry, aided sample preparation, filter, ip the filter, contaminant, eliminating detergent
Abstract
The Filter-Aided Sample Preparation (FASP) protocol enables efficient protein digestion and cleanup using membrane filters, eliminating detergents and contaminants prior to mass spectrometry.
Guidelines
Ensure all liquid has passed through the filter before proceeding to the next step.The membrane should remain visibly moist — it must have a wet sheen but never be completely dry. Drying can cause protein loss, membrane damage, or reduced recovery during subsequent washes and digestion steps.
Materials
Solutions:
DTT solution: 25 mM DTT in 50 mM ABC.
Precipitation buffer: 8M urea in 50 mM ABC
IAA solution: 55 mM IAA in 8M urea in 50 mM ABC.
ABC solution: 50 mM ABC
Materials:
FASP tubes: VN01H22 Sartorius
Troubleshooting
Safety warnings
ABC must be made fresh the same day of the experiment.
Urea will precipitate if exposed to high temperatures and prevent carbamylation
Reduction/Alkylation
1h 5m
Add DTT solution to the sample for a final concentration of 25 mM. Incubate at 80ºC for 15 min.
15m
Wait for the sample to cool down. Add urea to the sample to have a final concentration of 8M.
This can be either directly adding the urea powder to the solution, or diluting 5x with 8M urea in 50 mM ABC.
Spin sample through a 30k spin column until all the liquid goes through the filter, 15000r cf for 15.
15m
Add 100 µL of 55 mM IAA to the column. Incubate at Room temperature for 20 min in the dark.
20m
Spin through column at 15000 rcf for 15min.
15m
Wash
45m
Wash with 100 µL of 8 M urea in ABC. Spin through column at 15000 rcf for 15-20 min.
15m
Wash twice with 100 µL of ABC. Spin through column at 15000 rcf for 15min.
30m
Digestion
Make trypsin solution by adding 20 µL of 0.1 % TFA to a trypsin vial. Add 980 µL of ABC.
Place column into a new tube and add 100 µL of freshly prepared trypsin solution. Wrap the top of the tubes wit parafilm and incubate at 37 ºC O/N. Use a wet paper towel underneath the Eppendorf rack to minimise evaporation.