NEBExpress MBP Fusion and Purification System Quick Start Protocol (NEB #E8201)

  1. Subclone the gene of interest into the pMAL-c6T vector.

  2. Grow cells containing the pMAL-c6T fusion plasmid in LB containing ampicillin and 0.2% glucose to an A600 of ~0.5.

  3. Induce by adding IPTG to a final concentration of 0.3 mM.

  4. Grow for an additional 2 hours at 37°C, or 4 hours at 30°C, or 6–8 hours at room temperature, or overnight at 12–16°C.

  5. Harvest the cells and either freeze immediately or resuspend in 25 ml column buffer (CB) per liter of culture (CB, manual page 10).

  6. Lyse the cells by freeze-thaw followed by sonication.

  7. Clarify the lysate by centrifugation at 20,000 x g for 20 minutes.

  8. Dilute the supernatant (crude extract) by adding 125 ml cold CB for every 25 ml crude extract.

  9. Load the diluted crude extract on a 15 ml amylose column.

  10. Wash the column with ≥ 12 column volumes of CB.

  11. Elute the fusion protein with CB containing 10 mM maltose.