Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 184)

Appendix E
Sample Preparation
Preparing Lysates Prior to Purification
Lysates from E. coli cultures can be prepared using conventional sonication procedures
with the lysis buffers supplied in each kit, or using chemical lysis methods and the Profinia
bacterial lysis/extraction reagent. For
E. coli cultures expressing medium to high levels of
fusion proteins, (~10% of total protein), 200 ml of culture will normally 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.
Bacterial cultures can be grown in advance and centrifuged. The pellets can be stored at
–70°C for several months and lysed at a convenient date for sample preparation.
Native Lysate Preparation (Profinia Native IMAC or GST Kits)
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 native IMAC wash
buffer 1 or Profinia GST wash buffer (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).
4. Sonicate the lysate (on ice, using 25% output) four times at 1 min intervals.
5. Centrifuge at 16,000 x g for 20 min at 4°C to clarify the lysate.
6. Filter clarified lysate through a 0.45 µm filter to remove particulates.
7. Transfer the clarified lysate supernatant to a 15 ml or 50 ml sample tube and insert into
the Profinia instrument.
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 (Profinia Denaturing IMAC Kits)
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 denaturing IMAC
wash buffer 1 (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) four 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 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 the description under the native lysate prep for
treatment upon freezing/thawing.
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