Bio-Rad Bio-Gel P Polyacrylamide Gel - Manual (Page 17)

6. If the lysate is not going to be used immediately, it can be frozen at
–20°C and thawed once to be purified at a later date. See description
under the native lysate preparation (Profinia native IMAC or GST kits)
for treatment upon freezing/thawing.
Denaturing IMAC Lysate Preparation Using Bacterial
Lysis/Extraction Reagent
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 of Profinia
bacterial lysis/extraction reagent (Pierce catalog #78243 or 78266)
(200 ml of culture typically yields 0.8–1.0 g of paste or 8–10 ml of
lysate).
3. To decrease the viscosity, add a nuclease (DNase at 100 units/ml or
Benzonase at 25 units/ml) to the suspension, and thoroughly resuspend
by pipetting or vortexing. Let the solution incubate with gentle shaking
10 min at room temperature.
4. Centrifuge at 16,000 x g for 20 min at 4°C.
5. Discard the supernatant and add 10 volumes of Profinia denaturing
IMAC lysis buffer containing urea (see Table 3) to the inclusion body
pellet.
6. Vortex or mix well to thoroughly resuspend the pellet (may take ~10 min).
7. Centrifuge at 16,000 x g for 20 min at 4°C to clarify lysate.
8. Filter the clarified lysate through a 0.45 µm filter to remove particulates.
Transfer the clarified lysate to a 15 ml or 50 ml sample tube and insert
into the Profinia instrument.
9. If the lysate is not going to be used immediately, it can be frozen at
–20°C and thawed once to be purified at a later date. See description
under the native lysate preparation (Profinia native IMAC or GST kits)
for treatment upon freezing/thawing.
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