Igm Purification - GE HEALTHCARE AKTAprime plus Quick Reference Instructions

IgM purification

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, 0.8 M (NH
Elution buffer 1 (port B):
20 mM sodium phosphate, pH 7.5
Regeneration buffer 2 (port A2):
20 mM sodium phosphate, 30% isopropanol, pH 7.5
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 each inlet tubing from port A (8-port valve) in
eluents as given above and the tubing from port B
(2-port valve) in elution buffer 1.
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 15) 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 • p26
) SO , pH 7.5
4 2 4
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 IgM Purification HiTrap IgM
Purification.
Templates
Application Template
IgM Purification
HiTrap IgM Purification
c) Enter the sample volume and press
template.
IgM Purification HiTrap IgM Purification
Theoretical gradient in
Application Template.
%B
100
Priming
& Equilibration
50
Sample
Wash
11 10
Total separation time = 48 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
Elution Regeneration
Re-equilibration
11 11 5
00.0
Min