Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 206)

For additional cleaning-in-place options for eXact affinity and desalting cartridges, refer to
the Utilities section of the manual for information on the Affinity and Desalting Cartridge
Cleaning-In-Place method.
2.3 eXact Method Parameters and Instruction for Use
The default Bio-Rad eXact method requires the use of buffers at 1x concentrations which
are used by the preprogrammed methods stored on the instrument. The formulations pro-
vided in Table 2 list the recommended buffers for eXact purifications, as well as cleaning
and storage solutions designed for optimal regeneration and cartridge re-use. The concen-
tration of any buffer position used in the Profinia eXact method can be customized in the
Program Methods mode to 1x, 2x, 3x, 4x, and 5x. This option is particularly useful when
selecting 5 ml eXact purification methods with the 2 sample option, as the volume of buffer
required for some steps may exceed the capacity of standard bottles provided with the
instrument (~250 ml). In such cases, a custom eXact method may be programmed with
buffer concentration values between 2x–5x (the default is 1x) in order to minimized the
volume of buffer required by the method.
Optimal binding and elution settings for the eXact system are preset in the eXact method.
There are single wash and two wash method options. The two wash method option allows
the use of a second, optional buffer for increased wash stringency prior to elution. The
default Bio-Rad eXact methods allow three choices of incubation times, 0.5 hrs, 2 hrs, and
15 hrs. The optimal incubation time should be empirically determined and will be target
protein dependant; however, the cleavage reaction for most eXact fusion proteins occurs
within 30 min at room temperature. The incubation time can also be customized in the
Program Methods mode for durations outside the three standard options in the default
method.
Note: For cleavage reactions performed overnight, it is generally recommended to perform
the incubation at 4°C to minimize target protein degradation or denaturation. If the Profinia
instrument is stored and operated in a cold room, the cold room setting should be enabled
in the Utilities menu.
In general, binding/equilibration/wash buffers for eXact purifications should follow the rec-
ommendations found in Table 2 and the eXact System Manual. The recommended buffer
for desalting is the Bio-Rad Profinia desalting buffer (pH 7.4). When preparing for an eXact
purification, bottles should be clearly labeled with the buffer name and the buffer position
number (1–8) for insertion in the correct location on the instrument. The position number on
the bottle must match the position number on the instrument to properly run the prepro-
grammed methods. Follow the on-screen guide on the instrument for the correct set-up of a
eXact method and refer to the Quick Start Guide in Section 4 of the instrument manual for
detailed instructions. The sample load should be in the range of 2-50 ml; however, a minimum
load volume of 10 ml is recommended to avoid sample loss due to line priming and tubing
dead volumes. The recommended A
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for 1 mg/ml solution will be target protein dependant.
A typical run profile for 10 ml of Profinity eXact-tagged Maltose Binding Protein lyophilized
lysate (diluted 1:5 in 1x eXact Binding/Wash buffer) and using the recommended buffer
formulations in Table 2 is shown in Figure 1.
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