Purification Protocol
1. Equilibrate the cartridge with 10 column volumes
(CV) of bind/wash buffer at 3 ml/min (10 ml/min).
2. Load the sample lysate at 1 ml/min (5 ml/min).
3. Wash the cartridge with 10 CV of bind/wash
buffer at 3 ml/min (10 ml/min).
4. Equilibrate cartridge with 2 CV of elution buffer
at 3 ml/min (10 ml/min)*.
5. Stop flow and incubate for 30 minutes
**
.
6. Elute purified protein with 5 CV of elution buffer
at 3 ml/min (10 ml/min).
* Alternative elution protocol: Pump 5 CV of elution buffer at
0.1 ml/min (0.5 ml/min) at room temperature.
**Increased incubation time, 2–4 hr at 25°C or 12–24 hr at 4°C,
may be necessary for complete cleavage of some proteins.
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5.
Remove the supernatant, and filter through a
0.45 µM filter before applying to the cartridge.
Section 8
Cartridge Preparation and
Purification Protocol
To prepare a cartridge, remove the end plugs and
connect the cartridge to the chromatography system. The
cartridge is now ready for use. The following 1 ml
cartridge purification protocol is a general guideline
for first time users. Flow rates for 5 ml cartridges are
shown in parentheses. The kinetics of cleavage are
target protein specific and may require optimization
to maximize yield. See Figure 6 for a representative
purification profile using the following protocol. Refer
to the Profinity eXact System Manual for complete
details.
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