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.
- Vortex and thoroughly suspend magnetic beads.
- Aliquot 40 µl of bead suspension to a sterile microcentrifuge tube.
- 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.
- Add beads to 200-500 µl of cell culture extract.
- 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 MBP-fusion can be eluted from the beads or used directly for immunoprecipitation of target proteins.
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.