The protocol for sample cleanup using Millipore ZipTips can be found below, or downloaded as a pdf.
Solutions Required
Buffer A
100% Acetonitrile HPLC-grade
Buffer B
98% Milli-Q water
2% Acetonitrile HPLC-grade
0.1% Formic Acid (FA) for nanoLC or 0.1% Trifluoroacetic Acid (TFA) for MALDI
Buffer C
50% ACN
50% Milli-Q water
0.1% FA
Buffer D
Matrix CHCA or 2.5-DHB (concentration: 20 mg/mL)
-Dissolve 20mg of CHCA or DHB matrix in 1ml of 0.1 % of TFA in 30% of Acetonitrile
Procedure
Note: Resin bed provides backpressure, so set pipette to 10 μl, depress plunger to dead stop and slowly release.
1. Acidify sample (Vol 20-100 μl) by adding TFA (MALDI) or FA (nanoLC) to 0.1 % final concentration.
2. ZipTip equilibration
- Aspirate 10 μl Buffer A into tip and dispense to waste. Repeat.
- Aspirate 10 μl Buffer B into tip and dispense to waste. Repeat.
3. Bind and Wash the peptides/proteins
- Bind peptides to ZipTip pipette tip by aspirating and dispensing 3-7 cycles (simple mixtures), up to 10 cycles (complex).
- Aspirate 10 μl Buffer B and dispense to waste. Repeat wash once more.
4. Elution:
| nanoLC-MS |
MALDI-TOF/TOF |
Dispense 10μl of Buffer C into a clean Eppendorf tube.
Aspirate and dispense elution solution through ZipTip at least 5 times without introducing air.
Dispense 10μl of Buffer A into a clean Eppendorf tube.
Aspirate and dispense elution solution through ZipTip at least 5 times without introducing air.
Combined the elution solutions into an empty Eppendorf tube.
Dry in vacuum centrifuge.
Resuspend in 10 μl Buffer B |
Dispense 1-3μl of Buffer D into a clean Eppendorf tube.
Aspirate and dispense elution solution through ZipTip at least 5 times without introducing air. |
| Transfer to a recovery vial |
After 5 elution times through ZipTip spot on MALDI plate. Leave to dry |
