Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 201)

Appendix J
Profinia Profinity eXact Method Manual
Section 1
1.1 Background Information
The Profinia protein purification system is an automated, user-friendly instrument for the
purification of affinity tagged proteins and antibodies. The system uses standard
chromatography buffers, resins, and reagents along with the easy to use pre-programmed
purification methods stored on the instrument. There are six main types of applications
currently available in the Profinia system: native IMAC, denaturing IMAC, GST, desalting,
Protein A and G, and eXact. This section provides information on the use of the eXact
methods in the Bio-Rad Methods and Program Methods modes of the Profinia instrument.
1.2 Description of Method
The Profinia Profinity eXact method is designed for the affinity purification of tag-free target
protein from lysates prepared using the Profinity eXact
E. coli-based expression system
and purification cartridges. The instrument has two main types of eXact methods that can
be used for the purification of Profinity eXact affinity-tagged proteins: eXact affinity and
eXact plus integrated desalting. Both method types are designed for either 1 ml or 5 ml
pre-packed eXact cartridges and 10 ml or 50 ml P6 desalting cartridges (respectively) when
using the eXact plus desalting methods. Cartridges for eXact applications are
available from Bio-Rad as 1 ml and 5 ml eXact Bio-scale mini cartridges that are designed
for direct connection to the Profinia instrument. Each ml of Bio-Rad eXact resin typically
yields 2–3 mg of purified, tag-free target protein, depending on the concentration of the
target protein in the starting lysate.
The Profinia eXact methods allow selection of one or two samples per run, a choice of single
wash or two wash steps, and three preset incubation times for tag cleavage (0.5 hrs, 2 hrs,
and 15 hrs). In the Programed Methods mode, the incubation step can be set to any interval
up to 999 hrs. The eXact method also allows manual completion of the incubation step, at
the users discretion, advancing the program to the protein elution step. Following system
priming and cartridge equilibration, the eXact method is programmed to load sample from
the S1 and/or S2 ports onto the cartridge. The available pre-programmed Bio-Rad eXact
methods are designed for loading volumes between 2–50 ml per sample; however, the
recommended minimum load volume is 10 ml. For larger sample volumes, the Program
eXact methods can be customized to load in (ml) or (L) volumes ranging between 0–999.
Refer to Section 11 for system requirements when using large sample or buffer volumes on
the Profinia instrument. Table 1 summarizes the method steps for a typical Bio-Rad
Profinia 1 ml eXact + 10 ml Desalting Method with 1 sample. The steps outlined in Table 1
are similar for the 5 ml eXact methods; however, flow-rates are increased to accommodate
the larger volumes of the 5 ml eXact and 50 ml desalting cartridges. For eXact affinity only
methods (without integrated desalting), the steps associated with the desalting cartridge
are absent, and elution of the eXact cartridge is collected directly into 1D (or 2D) fractions
without desalting.
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