Affinity Chromatography - Department of Molecular and Cellular ...

The maximum recommended sample volume is 1.5 ml. See Table 17 for the effect of reducing
the sample volume applied to the column.
Table 17. Recommended sample and elution volumes using a syringe or Multipipette.
Sample load ml Add bufferml Elute and collect ml Yield % Remaining salt % Dilution factor
0.25 1.25 1.0 > 95 0.0 4.0
0.50 1.0 1.5 > 95 < 0.1 3.0
1.00 0.5 2.0 > 95 < 0.2 2.0
1.50 0 2.0 > 95 < 0.2 1.3
A simple peristaltic pump can also be used to apply sample and buffers.
Alternative 2: Simple desalting with ÄKTAprime
ÄKTAprime contains pre-programmed templates
for individual HiTrap Desalting 5 ml and
HiPrep 26/10 Desalting columns.
Buffer Preparation
Prepare at least 500 ml of the required buffer.
1. Follow the instructions supplied on the ÄKTAprime cue card to connect the column and load the system
with buffer.
2. Select the Application Template.
3. Start the method.
4. Enter the sample volume and press OK.
Figure 76 shows a typical procedure using ÄKTAprime. The UV (protein) and conductivity
(salt) traces enable pooling of the desalted fractions.
0.15
A 280 nm
Sample: (His) 6 protein eluted from
–– UV 280 nm
–– Conductivity
(His) protein
6
0.10
0.05
Salt
Column:
Buffer:
HiTrap Chelating HP with
sodium phosphate 20 mM,
sodium chloride 0.5 M,
imidazole 0.5 M, pH 7.4
HiTrap Desalting 5 ml
Sodium phosphate 20 mM,
sodium chloride 0.15 M, pH 7.0
Inject
0
0
1
2 min
Fig. 76. Desalting of a (His) 6
fusion protein on ÄKTAprime.
136