Bio-Rad Profinia™ Protein Purification Instrument - Manual

Automated Desalting of Proteins With the Profinia ™ Protein
Purification System: Comparison to Manual Desalting by Dialysis
Desalting With the Profinia System
To desalt a sample of BSA by gel filtration, the appropriate
method (either “10 ml Desalting” or “50 ml Desalting”,
depending on the sample size) was selected from the menu
of Profinia chromatography methods. The required reagents,
sample loop, and Bio-Scale
™ Mini Bio-Gel ® P-6 desalting
cartridge were installed as instructed by the instrument display
chromatography
tech note 5539
Shane Petersen and Laurie Usinger, Bio-Rad Laboratories, Inc.,
Hercules, CA 94547 USA
Introduction
Protein desalting is the process of separating proteins in a
solution from low molecular mass components. Desalting can
be used for buffer exchange, to remove low molecular mass
reagents, and to adjust the ionic strength of a protein solution.
Two commonly used methods for desalting are dialysis and gel
filtration. Dialysis uses a membrane to retain large molecules,
such as proteins, while allowing diffusion of smaller molecules
into a solvent. Gel filtration uses size exclusion to separate
proteins from small molecules: Small molecules permeate
the pores of the gel filtration media and are retarded, while
proteins larger than the molecular weight cutoff are excluded
from the pores and elute first.
The Profinia protein purification system is an automated
low-pressure chromatography system that can be used to
desalt proteins by gel filtration. Optimized methods are
preprogrammed and can be used with the buffers and
prepacked cartridges available in Profinia purification kits
(Figure 1). The preplumbed system is also easily maintained
through automated self-cleaning protocols. When used with
Profinia software, the system displays run data in real time
and allows the generation and printing of reports containing
chromatograms, method steps, and run information.
The present study compares the performance of the Profinia
system to dialysis, the most commonly used method for
protein desalting. Performance parameters tested include
the time required for complete desalting, exchange buffer
consumption, product yield, and sample dilution.
Methods
Bovine serum albumin (BSA) fraction V (Sigma-Aldrich) was
dissolved in 300 mM KCl and 50 mM potassium phosphate
to a final concentration of 3 mg/ml in preparation for desalting.
A 2 ml sample and a 10 ml sample were then desalted
using the Profinia system or by dialysis. The progress of
desalting was followed by measuring sample conductivity,
and protein concentration and yield were determined
spectrophotometrically using the sample absorbance at
280 nm and the known absorbance of a 1 mg/ml BSA
solution. Three desalting runs were performed with each method.
Fig. 1. Workflow from protein sample to desalted protein using the
Profinia system.
Buffer Preparation
Desalting Kit
Profinia Desalting Setup
Desalting
Data Analysis
Profinia Desalting Chromatogram