Bio-Rad Nuvia™ IMAC Resin - Manual (Page 13)

Bio-Scale Mini Nuvia IMAC Ni-Charged 9
Section 7
Preparation of E. coli Lysates
For E. coli cultures expressing medium to high levels of histidine-
tagged proteins (≥10% of total protein), 200 ml of culture will yield
sufficient material for a 1 ml cartridge purification, and 1,000 ml of
culture will yield sufficient material for a 5 ml cartridge purification
run. For cultures expressing protein at low levels (~10% of total
protein), the culture volumes will need to be determined empirically
for each protein.
Native lysates
1. Harvest cell pellet by centrifugation at 8,000 x g for 10 min at
4°C.
2. Determine weight of pellet and resuspend in 10 volumes native
lysis/wash buffer 1 (200 ml of culture typically yields 0.8 g of
paste and results in 8 ml of lysate).
3. Sonicate the lysate on ice four times at 1 min intervals.
4. Centrifuge the lysate at 12,000 x g for 20 min at 4°C.
5. Remove the supernatant and filter it through a 0.2 μm filter
immediately before applying to the cartridge.
Denatured lysates
1. Harvest cell pellet by centrifugation at 8,000 x g for 10 min at
4°C.
2. Determine weight of pellet and resuspend in 10 volumes
denaturing lysis/wash buffer 1 (200 ml of culture typically yields
0.8 g of paste, and results in 8 ml of lysate).
3. Sonicate the lysate four times at 1 min intervals.
4. Centrifuge the lysate at 12,000 x g for 20 min at 4°C.
5. Remove the supernatant and filter it through a 0.2 μm filter
immediately before applying to the cartridge.