BAF_Protocol_006 On-Bead Peptide Cleanup (Digested by Other Method)
Nicholas Sherman
Abstract
This protocol is for using beads as a cleanup step after protein is digested using a different method (not directly on Sera-Mag beads), for example solution digest. The peptides are precipitated onto the beads to allow for removal of salts and detergents that will interfere with downstream mass spectrometry.
Steps
On-Bead Clean-up
Stock Solution of magnetic Sera-Mag beads is made in section 1 of BAF_Protocol_004 and can be stored at 4C indefinitely and used as needed (never freeze).
Peptide digest (for example from a solution or in-gel digest) should be reduced in volume so that a 95% ACN (acetonitrile) final concentration can be made within the Eppendorf tube volume. This will precipitate the peptides onto the beads for clean-up. See next steps.
To the peptide digest that has been reduced in volume, add 2uL of bead stock mix (20ug).
Add 100% ACN to a final concentration of 95% ACN. Incubate for 8 minutes at room temperature.
Placed tubes on a magnetic rack for 2 minutes. Remove and discard supernatant.
Rinse beads with 180uL of 100% ACN, incubate for 30s then remove and discard supernatant.
Add 10uL of 2% DMSO in water. Give quick spin (2-3s) in bench-top centrifuge to aid liquid removal from tube walls to bottom of tube.
Place tubes in magnetic rack for 2 minutes and recover supernatant making sure not to recover any beads.
Dilute samples with 0.1% FA (formic acid in water) to <1% DMSO. Further clean-up using C18 tips may be done using BAF_Protocol_003.
