The following protocol is for the isolation of CBD-fusion protein from 200-500 µl cell culture supernatant.
CBD Column Binding Buffer
- Vortex and thoroughly suspend magnetic beads.
- Aliquot 50 µl of bead suspension to a sterile microcentrifuge tube.
- Add 500 µl CBD column buffer* and vortex to suspend. Apply magnet for 30 seconds, to pull beads to the side of the tube and decant supernatant. Repeat wash.
- Add 200-500 µl of cell culture supernatant to beads.
- Mix thoroughly and incubate at 4°C with agitation for 1 hour.
- Apply magnet and decant supernatant.
- Wash beads three times as in step 3 above.
At this point the purified CBD-fusion can be eluted from the beads or used directly for capture of target proteins.
CBD-Fusion Cleavage: To determine the required method of intein-fusion cleavage please refer to applicable NEB IMPACT Manual.
Notes: Efficiency of elution can be checked by eluting any protein that remains bound to the chitin magnetic beads with 50 µl of SDS-PAGE gel loading buffer and running 15 µl on a denaturing protein gel.
* 1X CBD Column Binding Buffer:
NaCl 500 mM, Tris-HCI 20 mM, EDTA 1 mM, Tween-20 0.1%, pH 8 @25°C.