Small Scale Affinity Chromatography (NEB #E8201)
To test multiple different clones to vary induction conditions for expression of a soluble fusion protein it is often easiest to use Amylose Magnetic Beads (NEB# E8035) to isolate the fusion protein. The binding capacity of these beads is 10 μg of fusion protein per mg of amylose magnetic beads. The following protocol is for the isolation of MBP-fusion protein from 200-500 μl cell culture supernatant.
MBP Column Binding Buffer
20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM DTT (optional), pH 7.5.
- Vortex and thoroughly suspend Amylose magnetic beads.
- Aliquot 100 μl of bead suspension to a sterile microcentrifuge tube.
- Add 500 μl of MBP column buffer and vortex to suspend. Apply magnet for 30 seconds to pull beads to the side of the tube and remove 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 remove supernatant.
- Wash beads three times as in Step 3 above.
- The purified MBP-fusion protein can now be eluted from the beads or used directly for capture of target proteins.
- Add 50 μl of MBP column buffer containing 10 mM maltose (elution buffer) to the bead pellet, vortex and incubate for 10 minutes at 4°C with agitation.
- Apply magnet and pipet eluted MBP-fusion protein into a clean microcentrifuge tube.
- Add an additional 50 μl of elution buffer to the beads and repeat elution step. Pool elution supernatants.
Note: Efficiency of elution can be checked by eluting any protein that remains bound to the Amylose Magnetic Beads with 50 μl of SDS-PAGE gel loading buffer and analyzing 15 μl by SDS-PAGE.