Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 202)

Table 1. Method Steps for Bio-Rad Profinia 1 ml eXact plus 10 ml Desalting Method
with 1 Sample
1.3 Required Equipment and Materials
Bio-Rad Profinity eXact 1 ml and 5 ml Bio-Scale Mini cartridges are designed to be used
with the pre-programmed eXact methods on the instrument. Profinity eXact methods that
include integrated desalting require the use of Bio-Gel P6 Desalting cartridges, available in
10 ml and 50 ml sizes, to match the corresponding 1 ml and 5 ml affinity cartridge sizes
required in the pre-programmed eXact methods. Complete Profinia buffer and purification
kits specific for eXact methods are not available; however, Table 2 lists the most common
recommended buffers and reagents used for eXact purifications. All buffers and solutions
can be made or purchased as concentrates as described in Table 2. Triggering anions
should be avoided in buffers used to prepare crude lysates and for the binding and wash
steps buffers. A list of triggering anions and buffer and chemical compatibilities are listed in
Tables 3 and 4.
Prepared buffers should use high quality, chromatography grade reagents and should be
pre-filtered through a 0.2 or 0.45 µm filter to remove particulates prior to use with the system.
Although the buffer concentrations are preset to 1x in the Bio-Rad eXact methods, buffer
concentrations of 1x, 2x, 3x, 4x, and 5x can be selected and saved as an alternate eXact
method in the Program Method mode on the Profinia instrument.
Note: Due to the larger volumes required (>250 ml) for some configurations of the 5 ml
eXact methods, external containers may be required for some buffers when the eXact
methods with the 2 sample options are selected. In such cases, external buffer containers
may be used to accommodate the larger buffer volumes required, or the eXact method
may be customized in the Program Method mode to select buffer concentrates (automatic
dilution) for any buffer position required for the method.
Accessories to the Profinia instrument that can be used for eXact methods include Profinia
cooling blocks and external sample/buffer tubing. Complete catalog and ordering
information can be found in Section 4 of this appendix.
Method Steps Buffer Name
Buffer
Port
Inlet
Buffer
Conc.
Fraction Port
Outlet
Column
Volume Flow Rate
[ml/min]
eXact
Cart.
Desalting
Cart.
System Priming All Buffer and Sample Ports
B0,1,3,4,5,6,7,8,
9 - Waste (W) - 20 Offline Offline
Equilibrate Di C2 Water B0 - W 2 4 Offline Online
Equilibrate C2 Desalting Buffer B4 1x W 3 4 Offline Online
Water Wash Water B0 - W - 20 Offline Offline
Equilibrate C1 eXact Equilibration/Wash Buffer B1 1x W 10 2 Online Offline
Load S1 to C1 Sample B9 1x W->1A 2-50 (ml) 1 Online Offline
Wash-1 C1 eXact Equilibration/Wash Buffer B1 1x 1A->1B 14 3 Online Offline
Prime Elute eXact Elution Buffer B3 1x W 4 10 Offline Offline
Pre-Elute C1 eXact Elution Buffer B3 1x W 1 3 Online Offline
Incubate - B0 - Inc.
Timed (0.5, 2,
or 15 hrs) 0 Offline Offline
Elute 1-C1 eXact Elution Buffer B3 1x W Up to 1 Online Offline
Elute 2-C1 eXact Elution Buffer B3 1x W 2.5 1 Online Online
Elute 1 C2 Desalting Buffer B4 1x W Up to 1 2 Offline Online
Elute 2 C2 Desalting Buffer B4 1x 1D 0.4 2 Offline Online
Elute 3 C2 Desalting Buffer B4 1x W 1 2 Offline Online
Clean 1 C2 Clean 1 Buffer B5 1x W 1 4 Offline Online
Clean 2 C2 Regeneration Buffer B6 1x W 2 4 Offline Online
Clean 1 C1 eXact Equilibration/Wash Buffer B1 1x W 3 3 Online Offline
Clean 2 C1 Regeneration Buffer B6 1x W 3 3 Online Offline
Clean 3 C1 eXact Equilibration/Wash Buffer B1 1x W 5 3 Online Offline
Water Wash Water B0 - W 0 20 Offline Offline
Clean 3 C2 Water B0 W 2 4 Offline Online
Store C1 eXact Storage Solution B7 1x W 7 2 Online Offline
Store C2 eXact Storage Solution B7 1x W 2 4 Offline Online
Water Wash Water B0 - W 4 20 Offline Offline
Clean Pump Seals Water B0 - W 4 20 Offline Offline
System Line
Cleaning Water
B0,
B9,
B10 -
W, 1A-D, 2A-
D - 20 Offline Offline
20% EtOH Wash 20% EtOH B8 1x W - 15 Offline Offline
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