Histidine-Tagged Protein Purification, Step Elution - GE HEALTHCARE AKTAprime plus Quick Reference Instructions

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
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.
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
prime should be displayed.
b) Use the arrow and OK buttons to move in the menu
tree until you find
Templates
Application Template
Affinity Purification
any HiTrap
c) Enter the sample volume and press
template.
Note: If a 5 ml column is preferred, see cue card on
p.36.
Theoretical gradient in
Template.
%B
100
Priming
Equilibration
50
and
**
1 10
Total separation time = 47 min + sample application time
Affinity Purification any
Run Application Template
Press OK to start
Run data displayed
Affinity Purification any HiTrap
Elution
Water wash &
priming
Sample
20
Controlled By:
HiTrap.
Set Sample Inj. Vol
(00.0 ml) 00.0
OK to start the
Application
Re-equili-
bration
10 5 Min