Apr 21, 2026
C18 and Resuspension (LTP; tube format)
This protocol is a draft, published without a DOI.
- 1Discovery Research Platform for Hidden Cell Biology
Protocol Citation: Georg Kustatscher 2026. C18 and Resuspension (LTP; tube format). protocols.io https://dx.doi.org/
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: In development
We are still developing and optimizing this protocol
Created: June 06, 2024
Last Modified: April 21, 2026
Protocol Integer ID: 101316
Keywords: C18, c18 procedure for desalting, c18 procedure, chromatin enrichment sample, tube format, hydrophobic contaminant, depletion of hydrophobic contaminant, whole cell lysates in low throughput, following sample clean, c18, contaminant, whole cell lysate, sample clean, desalting
Abstract
C18 procedure for desalting and depletion of hydrophobic contaminants for whole cell lysates in low throughput, tube format, following sample clean-up. For chromatin enrichment samples or high throughput processing, see alternative protocols.
Guidelines
Note: times are suggested and will depend on temperature and construction of C18. If necessary, increase time to ensure no liquid remains above the C18 at each step (but not too long to prevent C18 drying). Empty waste tube when required to ensure C18 tip does not touch flowthrough.
C18 construction
Insert 3 C18 discs into a p200 pipette tip using a plunger
- A maximum of 30ug can be loaded onto these tips
Insert tip into a 2ml tube with a hole in the top
- These waste tubes can be reused - keep separate from S-Trap waste tubes
C18 protocol
Wet the C18 tip using 90ul MeCN, centrifuge 500 rcf 2 min (# Need to do quickly after adding MeCN. As the solvent activates C18 and don’t want it to dry out
Equilibrate using 200ul 0.1% TFA, centrifuge 500 rcf 4 min
Acidify your sample by adding 10% TFA so that the final concentration is 1% (# Usually do this step first). (# we want to make 1% TFA in final volume. Also add without touching the sample and briefly centrifuge)
Load sample to C18, centrifuge 500 rcf until through (# Start with 4 minutes for 200 ul)
Wash with 200ul 0.1% TFA, centrifuge 1000 rcf 3 min
Elute sample into a new lobind tube using 25ul 66% MeCN in 0.1% TFA (2 parts MeCN, 1 part 0.1% TFA), centrifuge 500 rcf 1 min
- Can also elute directly into MS plate using a p200 pipette
Dry down sample in vacuum centrifuge (V-AQ setting - make sure fume hood is on and book!)
Can move to MS plate before drying down or after resuspending
- This can be at 30°C to speed up drying, going above this temperature will warp MS plate
Proceed to sample resuspension or store at 2-8°C
Sample resuspension
Resuspend in 0.1 % TFA to 0.5ug/ul
- Concentration depends on how much you want to inject and max injection vol
- If your sample is not already in a MS plate, transfer to one now
Add wash to H12 -> 40ul MeCN and 60ul 0.1% TFA
Spin down briefly to ensure all liquid is at the bottom of the well and to remove bubbles
Queue to LC-MS/MS