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

MAb purification, step elution

1
Preparing the buffers
•
Use high purity water and chemicals.
•
Filter all buffers thtough a 0.45 µm filter before use.
Binding buffer (port A1):
20 mM sodium phosphate, pH 7.0
Elution buffer (port B):
0.1 M glycine-HCl, 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 with 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 10) 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 • p22
*
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 Step
Templates
Application Template
Mab Purification
Step 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 Step Elution
Template.
%B
100
Priming
& Equilibration
50
Sample
Wash
11 10
Total separation time = 37 min + sample application time
Controlled By:
Elution.
Set Sample Inj. Vol
(00.0 ml) 00.0
Run Application Template
Press OK to start
Run data displayed
OK to start the
Application
Elution
Re-equilibration
11 6 Min