SPE Protein/Peptide Clean-Up Protocol
PREPARATION NOTES
The maximum amount of protein/peptides to load on the SPE Cartridge is 50 µg.
Reagents required in this protocol for bottom-up workflows:
acetonitrile, 0.1 % TFA, 5% acetonitrile/0.1% TFA, 50% acetonitrile/0.1% TFA
Reagents required in this protocol for top-down workflows:
acetonitrile, 0.1% TFA, 5% acetonitrile/0.1% TFA, 30% isopropanol/42% formic acid, 40% isopropanol/36% formic acid
PROTOCOL
- The SPE Cartridge must first be primed with the attached Priming Cartridge.
- Avoid excessive spinning, a few microliters of the final priming solution left behind is permissible during priming, loading, and wash. The final elution step can be spun to complete dryness.
- PRIME: Add 300 µL acetonitrile and spin through the SPE (2000 ×g (4500 rpm) ×2 minutes). Then add 300 µL 0.1% TFA (v/v) in water; spin (2000 ×g (4500 rpm) ×2 minutes).
- Discard the flow through solvents.
- Remove SPE Cartridge from the Priming Cartridge and attach to the base of the Filtration Cartridge containing the protein.
- LOAD: Spin the Filtration Cartridge with the SPE Cartridge attached at 2000 ×g (4500 rpm) ×5 minutes. Ensure that no more than a few microliters of solvent remains in the Filtration Cartridge, if so spin again. OPTIONAL: Reload the eluent into the SPE Cartridge (2000 ×g (4500 rpm) ×5 minutes). This second pass can improve SPE retention.
- WASH: Add 300 µL 5% acetonitrile, 0.1% TFA in water. Spin at 2000 ×g (4500 rpm) ×2 minutes. Discard solvent.
- ELUTE I: For digested peptides, add 300 µL of 50% acetonitrile, 0.1% TFA, water. Spin (2000 ×g (4500 rpm) ×5 minutes). Retain the eluent. OR
- ELUTE II: For intact protein, use 300 µL of 30% isopropanol/42% formic acid/28% water (2000 ×g (4500 rpm) ×5 minutes), followed by 300 µL of 40% isopropanol/36 % formic/24% water (2000 ×g (4500 rpm) ×5 minutes). Pool eluates.