Bio-Rad Bio-Gel P Polyacrylamide Gel - Manual (Page 16)

8. 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. However,
proteolysis or protein degradation can occur upon freezing and thawing,
and the quality of the purified product may be compromised. This will
have to be determined empirically for individual proteins. Upon thawing,
refilter through a 0.45 µm filter, as precipitates often form after freezing.
Denaturing Lysate Preparation
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
denaturing IMAC lysis buffer containing urea (see Table 3) (200 ml of
culture typically yields 0.8–1.0 g of paste or 8–10 ml of lysate).
Thoroughly resuspend the pellet by pipetting or vortexing.
3. Sonicate the lysate (on ice, using 25% output) 4 times at 1 min intervals.
4. Centrifuge at 16,000 x g for 20 min at 4°C to clarify lysate.
5. Filter clarified lysate through a 0.45 µm filter to remove particulates.
Transfer the filtered lysate to a 15 ml or 50 ml sample tube and insert
into the Profinia instrument.
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 the
description under the native lysate prep for treatment upon
freezing/thawing.
Native Lysate Preparation Using Bacterial Lysis/Extraction Reagent
(Recommended for IMAC procedures; binding capacities of GST fusion proteins will be
decreased ~30% using chemical lysis methodologies.)
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 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).
Thoroughly resuspend the pellet by pipetting or vortexing.
3. As an optional step and to decrease the viscosity, add a nuclease
solution (DNase at 100 units/ml or Benzonase at 25 units/ml) and
incubate for 10 min at room temperature.
4. Centrifuge at 16,000 x g for 20 min to clarify the lysate.
5. 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.
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