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  • Isolation of MBP-fusion protein using Amylose Magnetic Beads

    Introduction

    The following protocol is for the isolation of MBP-fusion protein from 200-500 µl cell culture supernatant.

    MBP Column Binding Buffer:
    200 mM NaCl
    20 mM Tris-HCl
    1 mM EDTA
    1 mM DTT
    (pH 7.4 @ 25°C)

    Protocol

    1. Vortex and thoroughly suspend magnetic beads.
    2. Aliquot 100 µl of bead suspension to a sterile microcentrifuge tube.
    3. 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 decant supernatant. Repeat wash.
    4. Add beads to 200-500 µl of cell culture supernatant to beads.
    5. Mix thoroughly and incubate at 4°C with agitation for 1 hour.
    6. Apply magnet and decant supernatant.
    7. Wash beads three times as in step 3 above.

      At this point the purified MBP-fusion can be eluted from the beads or used directly for capture of target proteins.

      MBP-Fusion Elution:

      1. 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.
      2. Apply magnet and pipet eluted MBP-fusion protein into a clean microcentrifuge tube.
      3. 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 running 15 µl on denaturing protein gel.