Before you start
- Acidify samples to pH < 3 (check with pH paper).
- Capacity depends on how many C18 disks are packed — check your supplier and add disks for larger amounts.
Reagents and materials
Totals are for 6 samples (+10% extra). Per-sample amounts are in the middle column.
| Reagent | Per sample | Total |
|---|---|---|
| Methanol or acetonitrile (activation) | 100 µL | 100 µL |
| Buffer B: 80% acetonitrile, 0.1% formic acid (conditioning + elution) | 150 µL | 150 µL |
| Buffer A: 0.1% TFA or 0.1% formic acid in water (equilibration + washes) | 300 µL | 300 µL |
| Acidified peptide sample (pH < 3) | 100 µL | 100 µL |
Equipment
- 200 µL pipette tips with 2–3 C18 disk plugs (or commercial C18 tips)
- Blunt needle for packing
- Centrifuge with tip adaptors
- Vacuum concentrator
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Activate
Pass 100 µL methanol or acetonitrile through the tip (centrifuge ~1,000–2,000 × g, 1–2 min).
Condition
Pass 50 µL buffer B.
Equilibrate
Pass 100 µL buffer A; do not let the disk dry out.
Load
Load the acidified sample slowly; reload the flow-through once if you want maximum recovery.
Wash
Wash twice with 100 µL buffer A.
Elute
Elute with 50–100 µL buffer B into a clean tube.
Dry and resuspend
Dry in a vacuum concentrator and resuspend in 2% acetonitrile / 0.1% formic acid for LC-MS/MS.
Critical points
- Samples must be acidic, or peptides will not bind.
- Keep the C18 wet between steps.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Low recovery | Check pH, avoid overloading, slow down loading, elute with higher acetonitrile. |
| Salt in MS | Add an extra wash. |
Safety
TFA, formic acid and acetonitrile: fume hood, gloves.
Further reading
- Rappsilber J et al. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nature Protocols (2007).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.