Removal of Endo F3 by Magnetic Chitin Beads
Endo F3 (NEB #P0771)
Chitin Magnetic Beads (NEB #E8036)
Magnetic Separation Rack (NEB #S1506, NEB #S1509)
- Pipette 50 µl chitin magnetic beads (NEB #E8036) into an eppendorf tube and place the eppendorf in a magnetic separation rack (NEB #S1506 or #S1509). Let the magnet attract the chitin beads, then pipette off the liquid supernatant and discard.
- With the eppendorf on the magnetic separation rack, wash the magnetic chitin beads 3 x 500 µl with 50 mM NH4 Formate pH 4.4 (or buffer of choice). Pipette of the supernatant and discard.
- Add the deglycosylated glycoprotein sample into the eppendorf with magnetic chitin beads.
- Rock the deglycosylated glycoprotein sample with the magnetic chitin beads for 10 minutes at 4°C.
- Place the eppendorf back on the magnetic separation rack, and allow the magnet to attract the chitin beads. Pipette off the supernatant and keep.
- Wash the magnetic chitin beads 3 x 100 µl with 50 mM NH4 Formate pH 4.4 (or buffer of choice). Pipette of the supernatant from each wash and keep.
- Combine all supernatants from Steps 5 & 6, as these are the deglycosylated glycoprotein.
- Analyze sample by method of choice
Notes on Use:
- Removal of Endo F3 from the deglycosylation reaction can be scaled up or down linearly with larger or smaller magnetic chitin bead volumes.
- The chitin magnetic beads binding capacity is 0.4 µg/µl of CBD-tagged protein. The concentration of Endo F3 Lot 1 is 0.32 µg/µl.