Mab Purification, Gradient Elution - GE HEALTHCARE AKTAprime plus Quick Reference Instructions

MAb purification, gradient 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):
100 mM sodium phosphate, 100 mM sodium citrate,
pH 7.0
*
Elution buffer (port B):
100 mM sodium phosphate, 100 mM sodium citrate,
pH 3.0
Prepare at least 500 ml of each eluent.
* With some antibodies, e.g. mouse IgG
to add NaCl up to 3 M in the binding buffer, to achieve efficient
binding when using HiTrap Protein A HP and HiTrap rProtein A FF.
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.
11-0027-48 AC, 2007-09 • p24
it might be necessary
1
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 Mab Purification Gradient
Templates
Application Template
Mab Purification
Gradient Elution
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 Mab Purification Gradient Elution
Application Template.
%B
100
Priming
& Equilibration
50
Sample
Wash 1
11 10
Total separation time = 63 min + sample application time
Controlled By:
Set Sample Inj. Vol
(00.0 ml)
Run Application Template
Press OK to start
Run data displayed
OK to start the
Wash 2
Elution
Re-equilibration
20 17 5
Elution.
00.0
Min