Removal Of Trypsin-Like Serine Proteases - GE HEALTHCARE AKTAprime plus Quick Reference Instructions

Removal of trypsin-like serine proteases

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):
50 mM Tris-HCl, 0.5 M NaCl, pH 7.4
Elution buffer (port B):
50 mM glycine-HCl buffer, pH 3.0
Prepare at least 500 ml of each eluent.
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.
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
(minimum 40) and position the white plate on the
fractionation arm against the first tube.
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
11-0027-48 AC, 2007-09 • p30
*
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
c) Enter the sample volume and press
template.
Note: If a 5 ml column is preferred see cue card on
p.36.
Templates
Application Template
Affinity Purification
any HiTrap
Affinity Purification any HiTrap
Theoretical gradient in
Template.
%B
100
Priming
Equilibration
Water wash &
priming
50
Sample
1 10
Total separation time = 47 min + sample application time
OK to start the
Set Sample Inj. Vol
(00.0 ml)
Run Application Template
Press OK to start
Run data displayed
Elution
Re-equili-
bration
20 10 5
HiTrap.
00.0
Application
Min