Home FAQs How do I eliminate Endo F3 from a reaction?

FAQ: How do I eliminate Endo F3 from a reaction?

Endo F3 can be removed from a deglycosylation reaction using NEB’s magnetic chitin beads.  Typical reaction conditions using magnetic chitin beads are as follows:

Materials:
Endo F3 (NEB #P0771 )
Chitin Magnetic Beads (NEB #E8036)
Magnetic Separation Rack (NEB #S1506, NEB #S1509)
  1. Pipette 50µl chitin magnetic beads ((NEB #E8036) into an eppendorf tube and place the eppendorf in a magnetic separation rack (NEB #S1506 or #S1509S).  Let the magnet attract the chitin beads, then pipette off the liquid supernatant and discard.
  2. With the eppendorf on the magnetic separation rack, wash the magnetic chitin beads 3 x 500uL with 50 mM NH4Formate pH 4.4 (or buffer of choice).  Pipette of the supernatant and discard.  
  3. Add the deglycosylated glycoprotein sample into the eppendorf with magnetic chitin beads.
  4. Rock the deglycosylated glycoprotein sample with the magnetic chitin beads for 10 minutes at 4°C.
  5. Place the eppendorf back on the magnetic separation rack, and allow the magnet to attract the chitin beads.  Pipette off the supernatant and keep.  
  6. Wash the magnetic chitin beads 3 x 100uL with 50 mM NH4Formate pH 4.4 (or buffer of choice).  Pipette of the supernatant from each wash and keep.  
  7. Combine all supernatants from steps 5 & 6, as these are the deglycosylated glycoprotein.
  8. Analyze sample by method of choice
Note - Removal of Endo F3 from the deglycosylation reaction can be scaled up linearly with larger magnetic chitin bead volumes.