Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 2)

© 2006 Bio-Rad Laboratories, Inc. Bulletin 5513
Results and Discussion
UV absorbance data from the Profinia purifications
demonstrate the performance of the automated GST +
Desalting method (Figure 2). The profiles of the UV traces at
the output of either column were virtually indistinguishable
from one another when compared using Profinia software.
The elution times for both the affinity and desalting peaks
varied by no more than 10 sec.
Fig. 2. Superimposed UV absorbance profiles from replicate GST + Desalting
purifications of the 51 kD protein on the Profinia system. The UV absorbance of
samples at 280 nm (AU) was collected over time for five consecutive purifications,
and the data are shown on the same pair of axes. (
— — ), absorbance at the output
of the affinity column; ( — — ), absorbance at the output of the desalting column.
Methods
A 51 kD GST-tagged protein expressed in Escherichia coli
was purified using both a Profinia system method and a
manual gravity-flow method for purification and desalting.
Five purification runs were performed, and samples of the
lysate, flow-through, wash, and purified protein fractions
from runs 1, 3, and 5 were then analyzed by SDS-PAGE.
The concentration of the purified 51 kD protein was determined
spectrophotometrically using the absorbance at 280 nm and
the known absorbance of a 1 mg/ml solution of the 51 kD
protein. Purity of the 51 kD protein was evaluated using the
Experion
™ automated electrophoresis system.
Protein Purification With the Profinia System
All purification reagents, columns, and a lyophilized E. coli
lysate containing the 51 kD GST-tagged protein were obtained
from the Profinia GST starter kit (catalog #620-0230). Each vial
of lysate was resuspended as directed in the kit instructions in
10 ml of Profinia lysis buffer. Purifications were performed with
the Profinia method template for “GST + Desalting” and with
the “low” sample flow rate. The 1 ml GST cartridge and 10 ml
desalting cartridge provided in the GST starter kit were used.
For each purification run, 5 ml of sample was loaded, which
required placement of 6 ml of lysate in the sample tube to
ensure complete loading of 5 ml of lysate. From each
purification run, 4 ml of purified and desalted protein was
collected. The five replicate runs were performed consecutively
using the same columns. Profinia software was used for real-
time monitoring of the purification runs and collection of the
subsequent run data.
Protein Purification With the Pierce GST Fusion Protein Purification
Kit and Zeba Desalt Spin Columns
Each vial of E. coli lysate containing the 51 kD GST-tagged
protein from the Profinia GST starter kit was dissolved in 10 ml
of the B-PER bacterial protein extraction reagent provided in the
B-PER GST fusion protein purification kit (Pierce Biotechnology).
Purifications were carried out according to the supplied
instructions using an application volume of 5 ml and an elution
volume of 4 ml. Eluted protein from each GST purification was
desalted using 10 ml Zeba desalt spin columns (Pierce
Biotechnology) following the manufacturer’s instructions.
Five purification runs were performed using new columns for
each replicate.
SDS-PAGE and Experion Pro260 Analysis
SDS-PAGE analysis was performed using the Criterion ™ system
and 8–16% Tris-HCl precast gels. Gels were stained with
Bio-Safe
™ Coomassie stain. The sample, flow-through, and
wash fractions were diluted 10-fold into Laemmli sample buffer
and loaded in a volume of 10 µl. Purified protein was diluted in
Laemmli sample buffer to 0.1 µg/µl and loaded in a volume of
10 µl (1 µg). Experion analysis of the purified protein fractions
was performed using the Experion Pro260 analysis kit
following the protocol described in the instruction manual.
20 25 30 35 40 45 50
Run time, min
AU
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Fig. 1. Example of a Profinia standard report. Profinia software prepares a report
containing chromatograms, continuous records of various method parameters,
protein yield, sample application and elution volumes, and purification conditions.
51 kD
affinity peak
51 kD
desalted peak