Oct 28, 2025
FASP (for fChEP samples)
- Victoria Munro1
- 1University of Edinburgh
Protocol Citation: Victoria Munro 2025. FASP (for fChEP samples). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rry6g1b/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 06, 2024
Last Modified: October 28, 2025
Protocol Integer ID: 101329
Keywords: ChEP, FASP, fchep sample, fasp, sample preparation, fchep, sample, preparation, containing sd, filter
Abstract
Filter aided sample preparation (FASP) clean-up protocol optimised for fChEP samples containing 1% SDS.
Guidelines
Day 1 spin time = 3 hours (+ 40 min for reduction and alkylation)
Day 2 spin time = 40 minutes
ABC per sample = 500ul
Urea/ABC per sample = 1ml
Materials
Vivacon 500, Hydrosart, 30,000 MWCO (APPLETON WOODS LTD, VN01H22)
Urea (Sigma-Aldrich, U5378-500G)
Ammonium bicarbonate (Sigma-Aldrich, A6141-500G)
Dithiothreitol (DTT; Promega, V3151)
Iodoacetamide (IAA; ?)
Trypsin (Thermo, 90058)
Troubleshooting
Safety warnings
Note: this protocol has 2 extra urea/ABC washes compared to the original and is for samples which have not yet been reduced and alkylated. The number of washes will be sample and buffer dependent and may need optimising.
Sample preparation
Start with cell lysate with sheared DNA and of known protein concentration.
Heat at 95°C for 5 min.
Spin at 16,000 x g for 5 min to clarify the lysate
Load to filter, wash with urea, reduction and alkylation
Combine pre-determined amount of sample with 8M urea, 50 mM ammonium bicarbonate in the 30kDA MWCO filter unit so that total volume is 200ul. Spin at 14,000 rcf for 20 minutes.
Add 200 μl 8M urea, 50 mM ammonium bicarbonate to filter, and spin at 14,000 rcf for 20 minutes. Discard flow through
Add 100 μl 10 mM DTT in 8M urea, 50 mM ammonium bicarbonate. Incubate at room temperature for 20 minutes
Then add 100 ul 55mM IAA (27.5 mM final) in 8M urea, 50mM ABC. Incubate in dark at room temperature for 20 minutes.
Spin 14,000 rcf for 20 minutes
Add 100 μl 8M urea, 50 mM ABC to filter. Spin at 14,000 rcf for 20 minutes
100 μl 8M urea, 50 mM ammonium bicarbonate, and spin again
100 μl 8M urea, 50 mM ammonium bicarbonate, and spin again
100 μl 8M urea, 50 mM ammonium bicarbonate, and spin again
Deplete urea
Add 100 μl of 50 mM ammonium bicarbonate. Spin at 14,000 rcf for 20 minutes
Repeat 100 μl 50 mM ammonium bicarbonate, and spin again
Digestion
Transfer the filter unit to a new collection tube
Add 1/20 trypsin in 100ul 50mM ABC to each sample. Parafilm tube lids shut. Digest 4 - 18 hrs (usually 16 h) in 37°C incubation hood with wet paper towel.
Peptide elution
Collect filtrate by spinning at 14,000 rcf for 20 minutes. If dried out, add 100ul 50mM ABC first. (Instead of 100 ul add 50 ul of 50 mM ABC so that the final volume isn’t that high).
Add 100uL of ABC and spin again for 20min @ 14,000 rcf. Collect again.
Proceed to C18