GE HEALTHCARE AKTAprime plus, Quick Reference Instructions

Page: 8 / 40

11-0027-48 AC, 2007-09 • p8
e) Connect a sample loop large enough for your sample
between port 2 and 6 on the injection valve. Use a
syringe to manually fill the loop.
Note: If a Superloop is needed, additional information
is supplied in the instructions for Superloop.
4
Selecting Application Template and
starting the method
a) Check the communication to PrimeView. At the lower
right corner of the screen the text Controlled By:
prime should be displayed.
b) Use the arrow and OK buttons to move in the menu
tree until you find Affinity Purification any HiTrap .
Histidine-tagged protein purification, step
elution
1
Preparing the buffers
•
Use high purity water and chemicals.
•
Filter all buffers through a 0.45 µm filter before use.
Binding buffer (port A1):
20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole,
pH 7.4
*
Elution buffer (port B):
20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole,
pH 7.4
Prepare at least 500 ml of each eluent.
* Alternative binding buffers:
5–40 mM imidazole can be included in the binding buffer to
reduce unspecific binding of non-histidine-tagged proteins.
The concentration of imidazole is protein dependent and if the
protein of interest elutes or does not bind at a certain imidazole
concentration, then reduce the concentration.
2
Preparing the sample
a) Adjust the sample to composition of binding buffer by:
•
diluting the sample in binding buffer
or
•
by buffer exchange using HiTrap Desalting or HiPrep
26/10 Desalting.
b) Pass the sample through a 0.45 µm filter.
Note: If HisTrap FF crude column is used no filtration
nor clarification of the sample is needed.
3
Preparing the system
a) Place the inlet tubing from port A1 (8-port valve) in
the binding buffer and the tubing from port B (2-port
valve) in the elution buffer.
b) Place the three brown waste tubings in waste.
c) Connect the column between port 1 on the injection
valve (7-port valve) and the UV flow cell (see Ordering
information on next page for suitable columns).
d) Fill the fraction collector rack with 18 mm tubes
**
and
position the white plate on the fractionation arm
against the first tube.
** The number of tubes to insert in the fraction collector varies with
the sample volume. Fill the fraction collector with 20 tubes + one
tube/ml sample. For example, if the sample volume is 10 ml, fill
the fraction collector with 20 + 10 = 30 tubes. However, note
that the maximum capacity of the fraction collector is 95 tubes,
limiting the sample volume to 75 ml.
Theoretical gradient in Affinity Purification any HiTrap Application
Template.
Affinity Purification
any HiTrap
Set Sample Inj. Vol
(00.0 ml) 00.0
Run Application Template
Press OK to start
Run data displayed
Application Template
Templates
1 10 20 10 5 Min
100
50
Sample
Priming
Elution
Total separation time = 47 min + sample application time
Water wash &
priming
%B
Re-equili-
bration
Equilibration
c) Enter the sample volume and press OK to start the
template.
Note: If a 5 ml column is preferred, see cue card on
p.36.