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  • Isolation of MBP-fusion protein using Anti-MBP Magnetic Beads

    The following protocol is for the isolation of 2.5 µg of MBP-fusion protein from 200-500 µl cell culture extract at a concentration of 1 mg/ml extract.

    1. Vortex and thoroughly suspend magnetic beads.
    2. Aliquot 40 µl of bead suspension to a sterile microcentrifuge tube.
    3. Add 500 µl 0.1 M NaPhosphate Buffer (pH 8.0) and vortex to resuspend. 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 extract.
    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 immunoprecipitation of target proteins.

      MBP-Fusion Elution:

      1. Resuspend bead pellet in 40 µl of 3 X SDS sample loading buffer [187.5 mM Tris-HCl (pH 6.8), 6% (w/v) SDS, 30% glycerol, 150 mM DTT, 0.03% (w/v) Bromophenol blue, 2% β-mercaptoethanol].

      2. Heat sample at 70°C for 5 minutes.

      3. Place sample tube on magnetic rack for 30 seconds; load 20 µl of supernatant on SDS-PAGE gel and electrophorese.

      4. Transfer to PVDF membrane and probe with appropriate antibody.