Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 3)

Both the automated and manual methods compared in this
study are capable of purifying the 51 kD protein to apparent
homogeneity, as determined by SDS-PAGE and Coomassie
Blue staining (Figure 3). Both the Bio-Scale
™ Mini GST
cartridges used with the Profinia system and the gravity-flow
affinity columns used with the manual method bound most
of the tagged fusion protein applied, as judged by comparing
the profiles of the flow-through fractions to those of the
unfractionated lysate. The protein profile in the flow-through
fraction and the purity of the protein product were consistent
from run to run with both methods tested (Figure 3).
Though both systems delivered 51 kD GST-tagged protein
of comparable purity, the Profinia system yielded slightly more
purified protein with less variability in yield than did the manual
system (Table 1). This increased reproducibility may be due to
the fact that the Profinia system reduces sample handling.
Table 1. Yield and purity of the 51 kD protein. Protein was purified and desalted
either with the Profinia system and GST starter kit, or manually with the B-PER GST
fusion protein purification kit and Zeba desalt spin columns. Average yield of the five
runs performed using each method is shown ± standard deviation.
Purification Method Average Yield (mg) Average Purity (%)
Manual system 4.34 ± 0.24 96.1
Profinia system 4.76 ± 0.14 96.2
Both purification methods required approximately 90 min to
complete. With the manual method, purified and desalted
protein was available in 90 min. However, the purified protein
from the Profinia system was available and ready for use in
downstream applications within 55 min for all five purifications.
The remaining time required for the Profinia system is
accounted for by the automated cycle for column cleaning and
regeneration, a step not carried out using the manual method.
The manual method required continuous user presence in
order to apply buffer and collect fractions, while the Profinia
© 2006 Bio-Rad Laboratories, Inc. Bulletin 5513
Fig. 3. SDS-PAGE analysis of purification fractions. Samples were separated by SDS-PAGE on 8–16% Tris-HCl Criterion precast gels, and gels were stained with Bio-Safe
Coomassie stain. A, samples from the Profinia separations; B, samples from the manual separations. Lane designations are as follows: M, markers (Precision Plus Protein
™
dual color standards); L, lysate (unfractionated); FT, flow-through fraction; W, wash fraction; P, purified protein.
system ran unattended and only required approximately
5 min of hands-on time to initiate and complete the
purification. The Profinia system automatically collected the
sample from the affinity column and applied it to the desalting
column. This must be performed manually in the gravity-flow
method in a step that also requires the use of a centrifuge.
In contrast to the Profinia system, the length of time using
the gravity-based purification varied by as much as 30 min due
to flow rate differences between the columns used in the
manual method.
Conclusions
The Profinia protein purification system has been tested
against a manual gravity-flow purification system for
purification of a GST-tagged protein. The manual system
used in this comparison matched the Profinia system in
application volume, column volumes, and elution volume.
The Profinia system is fully automated and requires no user
handling between application of the sample and conclusion of
both the affinity purification and desalting steps. While reduced
sample handling and hands-on time are the principal benefits
of the Profinia system over manual purification, the Profinia
system appeared superior when compared to the manual
method tested in terms of protein yield and reproducibility, and
equivalent in terms of protein purity.
A key feature of the Profinia system is automated collection
of purification data, an option not available with the manual
system, and this simplifies evaluation of the effectiveness of
purification. In addition, a report is prepared that contains
chromatograms, continuous records of various method
parameters, yield, volumes, and purification conditions
(Figure 1).
B. Manual system
Run 1 Run 3 Run 5
M L FT W P FT W P FT W P
A. Profinia system
Run 1 Run 3 Run 5
M L FT W P FT W P FT W P
MW, kD
250
150
100
75
50
37
25
20
15
10
MW, kD
250
150
100
75
50
37
25
20
15
10