Continue expression of GFP. Continue MBP "peak 4" purification using affinity chromatography.
- After overnight growth, spin cells at 4500rpm at 4°C for 15 minutes.
- Resuspend each of the pellets in 1mL of LB broth (taken from 1L sterile LB previously made in 2800mL flasks.
- Add each of the resuspended pellets with broth to one of the four 2800mL flasks previously made with 1L of sterile LB.
- Add 1mL 100mg/mL ampicillin to each flask.
- Incubate these four flasks at 37°C at 165rpm until 0.6 OD is reached.
- Add 1mL 0.1M IPTG to each flask and decrease temperature of growth to 25°C.
- Let the cells continue to grow and express protein overnight.
Purifying "Peak 4" of MBP
- Run 40mL column buffer (20mM Tris-HCl (pH 7.4), 200mM NaCl, and 1mM EDTA) at 1mL/min over the amylose resin column.
- Run 100mL of 10mM maltose in column buffer over the column at 1mL/min.
- 10mM maltose(100mL) = 0.36g maltose + column buffer