Oct 24, 2025
- Cristina CARDENAL PERALTA1
- 1University of Edinburgh
Protocol Citation: Cristina CARDENAL PERALTA 2025. FASP-No IP. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8d26l5r/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: 102301
Keywords: FASP, Mass Spectrometry, efficient protein digestion, fasp, membrane filter, using membrane filter, mass spectrometry, aided sample preparation, filter, no ip the filter, ip the filter, contaminant, eliminating detergent
Abstract
The Filter-Aided Sample Preparation (FASP) protocol enables efficient protein digestion and clean-up 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
Buffers:
Precipitation buffer: 8M urea in 50 mM ABC
DTT solution: 10 mM DTT in 8M urea in 50 mM ABC
IAA solution: 55 mM IAA in 8M urea in 50 mM ABC
ABC: 50 mM of ammonium bicarbonate
Materials:
FASP tubes: VN01H22 Sartorius
Troubleshooting
Safety warnings
Always avoid urea > 40 °C (prevents carbamylation).
Sample reception
15m
Combine 30
Spin at 15000 rcf for 15 min
15m
Add 200 µL of precipitation buffer to the filter unit. Spin at 15000 rcf for 15 min.
15m
Reduction and Alkylation
15m
If the sample was lysed without DTT, add 100 µL of DTT solution. Incubate at Room temperature for 20min.
20m
Add 100 µL of IAA solution. Incubate in the dark for 20 min.
20m
Spin at 15000 rcf for 15 min.
Washes
1h
Add 100 µL of precipitation buffer to filter, spin at 15000 rcf for 15 min.
Add 100 µL of 50 mM ABC to filter, spin at 15000 rcf for 15 min.
These washes serve the purpose of eliminating urea from the sample, as trypsin is not active in high urea concentrations.
15m
Repeat previous step 2 times. (If the sample was lysed with SDS, increase the number of washes by two or three).
45m
Transfer the filter unit to a new collection tube. Label both tube and filter.
Digestion
Add 20 µL of 0.1 % TFA to the trypsin vial, then add 980 µL of ABC.
Add 100 µL of the newly made trypsin solution to each filter unit. Incubate O/N at 37 ºC. Wrap each tube with parafilm and put a wet paper towel underneath the rack to ensure the incubation environment remains moist.
Collect filtrate by spinning at 15000rcf for 15 minutes.
Add 100ul of ABC and spin again for 15 min at 15000 rcf. Collect again.
Sample acidification
Add 20 µL of 10 % TFA to acidify the samples. Proceed to StageTips