Filter through 50 – 100 µL of methanol without letting all solution pass, keeping the membrane wet. Discard filtrate.

Repeat conditioning if membrane has gone dry.

For centrifugation processing we recommend 1,000 – 1,500 x g for all steps.

Filter through 50 – 100 µL of 0.5% trifluoroacetic acid without letting all solution pass, keeping the membrane wet. Discard filtrate.

Restart protocol at conditioning if membrane has gone dry.

Put StageTip into a new collection tube and pipette sample into StageTip. Maximum volume of 400 µL. 40 µg maximum recommended peptide mass per C18-HD StageTip.

Pass all solution through membrane and keep filtrate for troubleshooting problems.

Optionally, user may pass the sample filtrate through an additional time, which may improve retention.

Put StageTip into a new collection tube.

Filter through 50 – 100 µL of 0.5% trifluoroacetic acid.

Filter through 50 – 100 µL of 0.5% acetic acid.

Depending on the level of contaminants in the sample, more wash steps may be necessary.

Put StageTip into a new collection tube for your analyte.

Filter through 10 – 30 µL of 80% acetonitrile, 0.5% acetic acid, in water.

Repeat step 5b one to two more times to elute all the analyte.

Elution volume for high recovery may depend on how many micrograms of sample loaded. At maximum capacity, 100 µL total over a few elutions may be necessary.

The sample is clean and ready for further concentration followed by mass spectrometry analysis.