Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 185)

Native Lysate Preparation Using Profinia Bacterial Lysis/Extraction Reagent
(Recommended for IMAC procedures; binding capacities of GST fusion proteins will be
decreased ~30% using chemical lysis methodologies.)
1. Harvest the 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 (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 U/ml or benzonase at 25 U/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.
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 Profinia Bacterial Lysis/Extraction
Reagent
1. Harvest the cell pellet by centrifugation at 8,000 x g for 10 min at 4°C.
2. Determine weight of the pellet and resuspend in 10 volumes of Profinia bacterial
lysis/extraction reagent (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 U/ml or benzonase at 25
U/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 wash buffer 1
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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