Quick Start Guide (E8200)
- Subclone the gene of interest into the pMAL-5 vector of choice.
- Grow cells containing the pMAL-5 fusion plasmid in LB amp 0.2% glucose to an A600 of around 0.5.
- Induce by adding IPTG to a final concentration of 0.3 mM.
- 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.
- Harvest the cells and resuspend in 25 ml column buffer per liter of culture (CB, p.17).
- Lyse the cells by freeze-thaw followed by sonication.
- Clarify the lysed cells by centrifugation at 20,000 x g for 20 m.
- Dilute the supernatant (crude extract) by adding 125 ml cold column buffer for every 25 ml crude extract.
- Load the diluted crude extract on an amylose column.
- Wash the column with ≥ 12 column volumes of CB.
- Elute the fusion protein with column buffer + 10 mM maltose.