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

Life Science
Group
06-0784 0107 Sig 1106 Bulletin 5539 US/EG Rev A
Bio-Rad
Laboratories, Inc.
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Fig. 2. UV absorbance and conductivity profiles of desalting runs on the
Profinia system. Absorbance at 280 nm and conductivity readings of BSA
samples were collected over time. (
— — ), UV absorbance at 280 nm; ( — — ),
conductivity trace. The two vertical lines mark the collected protein peak (1D),
which is resolved completely from the salt peak.
screen. To desalt the 2 ml sample, a 2 ml sample loop and
a 10 ml desalting cartridge were used. To desalt the 10 ml
sample, a 10 ml sample loop and a 50 ml desalting cartridge
were used. Profinia software was used for real-time monitoring
of the UV absorbance and conductivity of the sample during
the desalting run and for collection and analysis of run data.
Buffers and desalting cartridges are part of the Profinia
desalting kit, while sample loops can be obtained as an
accessory for the desalting-only application.
Desalting by Dialysis
The dialysis protocol described by Zumstein (1994) was
followed using a Spectra/Por dialysis membrane with a
molecular weight cutoff of 14 kD (Spectrum Laboratories) and
a 1:500 volume of phosphate buffered saline (PBS). During
desalting, PBS was mixed continuously using a magnetic
stirrer, and conductivity of the BSA sample was monitored
at 15 min intervals using a MeterLab CDM210 conductivity
meter (Radiometer Analytical). Dialysis was complete when
the conductivity of the sample reached equilibrium with that
of the exchange buffer.
Results and Discussion
Using the Profinia system, UV absorbance and conductivity
data were automatically collected and plotted to demonstrate
the performance of this desalting method (Figure 2). The
Profinia system also automatically delivered the desalted
protein product to a collection tube in 15 min, regardless
of the sample size being desalted. In contrast, the dialysis
method required manual conductivity measurements to
determine when buffer exchange was complete.
Both methods were capable of desalting protein samples
to completion; however, there were notable differences in
performance (Table 1). The time required to complete desalting
with the Profinia system was 8- to 10-fold less than the time
required for dialysis, depending on the size of the sample.
Although both methods resulted in >90% yield, the final
concentration of the dialyzed protein sample was 2-fold higher.
Finally, the Profinia system used 20-fold less desalting buffer
than was required for dialysis.
Table 1. Performance comparison of the two desalting methods.
Average values from three replicate desalting runs (n = 3) are shown.
Final Protein
Total Time Concentration Recovery Buffer
Method Required (mg/ml) (mg) Yield Used
2 ml Sample
Profinia system
15 min 1.4 5.6 93% 50 ml
Dialysis 120 min 2.8 5.6 93% 1,000 ml
10 ml Sample
Profinia system
15 min 1.4 28.0 93% 250 ml
Dialysis 150 min 2.7 28.3 94% 5,000 ml
Conclusions
Dialysis and gel filtration chromatography are two commonly
used methods for desalting protein solutions. As the
mechanism of buffer exchange is not the same, there are
advantages to each method. Dialysis will usually produce a
more concentrated product than gel filtration, though some
sample dilution should still be expected. Desalting by gel
filtration using the Profinia protein purification system offers
substantial time savings as well as the significant advantages
of greatly decreased buffer consumption and an automated,
scalable process.
Reference
Zumstein L, Dialysis and ultrafiltration, appendix 3C.1 in Current Protocols
in Molecular Biology (Ausubel FM et al., eds), Wiley, New York (1994)
Information in this tech note was current as of the date of writing (2006) and
not necessarily the date this version (rev A, 2007) was published.
AU (UV channel 2)
Run time, min
0.4
0.3
0.2
0.1
0.0
12 13 14 15 16 17 18
2 ml BSA
Conductivity
, mS/cm
100
80
60
40
20
AU (UV channel 2)
Run time, min
0.4
0.3
0.2
0.1
0.0
12 13 14 15 16 17 18
10 ml BSA
Conductivity
, mS/cm
100
80
60
40
20
BSA
Salt
BSA
Salt
1D
1D