Bio-rad Nuvia™ IMAC Resin Uživatelský manuál stáhnout pdf (Strana 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.

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Bio-rad Nuvia™ IMAC Resin Uživatelský manuál Strana 13