Bio-Rad Bio-Gel P Polyacrylamide Gel - Manual (Page 15)

4. Return the 3-way stopcock lever to the middle position, facing toward
the syringe. The desalting loop is now filled with sample and ready for
the desalting method to begin.
Once installed, the cartridges are automatically equilibrated by the
preprogrammed methods. After use, the cartridges should be stored at
4°C and be used within 6 months.
Section 6
Sample Preparation
6.1 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
compatible chemical lysis reagents. 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 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 lysis buffer or Profinia GST lysis buffer depending on the
application (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).
4. Sonicate the lysate (on ice, using 25% output) 4 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 supernatant through a 0.45 µm filter to remove
particulates.
7. Transfer the filtered lysate to a 15 ml or 50 ml sample tube and insert
into the Profinia instrument.
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