11-0027-48 AC, 2007-09 • p26
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
4
)
2
SO
4
, pH 7.5
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.
IgM Purification
HiTrap IgM Purification
Set Sample Inj. Vol
(00.0 ml)
00.0
Run Application Template
Press OK to start
Run data displayed
Application Template
Templates
4
Selecting Application Template and
starting the method
a) Check the communication to PrimeView. At the lower
right corner of the screen the text
Controlled By:
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
.
c) Enter the sample volume and press
OK
to start the
template.
Theoretical gradient in
IgM Purification HiTrap IgM Purification
Application Template.
Re-equilibration
Wash
Elution
Priming
& Equilibration
Regeneration
100
50
11
10
11
11
5
Min
Sample
Total separation time = 48 min + sample application time
%B
Summary of Contents for AKTAprime plus
Page 39: ...11 0027 48 AC 2007 09 p39...
