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.