Quick Start Guide (E8200)

Protocol

1. Subclone the gene of interest into the pMAL-5 vector of choice.
2. Grow cells containing the pMAL-5 fusion plasmid in LB amp 0.2% glucose to an A₆₀₀ of around 0.5.
3. Induce by adding IPTG to a final concentration of 0.3 mM.
4. Grow for an additional 2 h at 37°C, 4 h at 30°C, 6-8 h at room temperature, or
   overnight at 12-16°C.
5. Harvest the cells and resuspend in 25 ml column buffer per liter of culture (CB, p.17).
6. Lyse the cells by freeze-thaw followed by sonication.
7. Clarify the lysed cells by centrifugation at 20,000 x g for 20 m.
8. Dilute the supernatant (crude extract) by adding 125 ml cold column buffer for every 25 ml crude extract.
9. Load the diluted crude extract on an amylose column.
10. Wash the column with ≥ 12 column volumes of CB.
11. Elute the fusion protein with column buffer + 10 mM maltose.