GE HEALTHCARE AKTAprime plus User manual

ÄKTAprime plus
GE Healthcare
Cue Cards
Contents
System Preparation 2
Buer exchange on HiTrap Desalting 4
Buer exchange on HiPrep 26/10 Desalting 6
Histidine-tagged protein purication,
step elution 8
Histidine-tagged protein purication,
gradient elution 10
IMAC purication - any metal 12
On-column refolding 14
GST-tagged protein purication 16
Strep(II)-tagged protein purication 18
MBP-tagged protein purication 20
MAb purication, step elution 22
MAb purication, gradient elution 24
IgM purication 26
Albumin removal 28
Removal of trypsin-like serine proteases 30
Anion exchange 32
Cation exchange 34
Method Templates value table 36

11-0027-48 AC, 2007-09 • p2
System Preparation
1 Starting the system
a) Press the button on the back of the system.
b) Wait until the system self-test has been completed
(30-40 seconds). Templates is displayed when the
system is ready to use.
2 Starting the computer
a) Start the computer.
b) Start PrimeView™.
c) Check the communication between system and
computer.
Tip! If communication has been established, the
text Controlled By: prime should be displayed at
the lower right corner of the screen. If not, check the
connections.
3 Purging the system ow path
a) If the ow path is lled with storage solution (e.g.
ethanol 20%), remove it as follows:
• Place the three brown waste tubings in waste.
• Set all inlet tubings (B, A1, …) that will be used in
water (see the appropriate cue card).
• Select the application template System Wash
Method, select the inlets to be washed. Press OK.
Note: Wait at least 15 minutes before using the
system after the lamp has been turned on to avoid
drifting UV base line.
c) If using pH:
Calibrate the pH. The pH must be calibrated every day.
See ÄKTAprime plus User Manual for instructions.
5 Checking the ow rate
a) Set the injection valve to position WASTE.
b) Start the ow (1-50 ml/min).
c) Use a graduated glass cylinder to collect at the WASTE
outlet (port 5). Collect during at least one minute.
Note: Inaccurate ow rate may be due to air in the
pump. If this is the case, ush the pump with buer
and try again. If the problem persists, ush the system
with ehtanol or methanol followed by water.
System Wash Method
Select Buffer V. Pos
B,A:_,_,_,_,_,_,_,_ OK
Application Template
Templates
b) If there are large amounts of air in the tubing:
Fill the tubing using Purge kit P-950. See ÄKTAprime™
plus User Manual for detailed instructions.
4 Preparing the monitors
a) Check the UV lamp lter position and the lamp
position.
b) Check that the UV lamp is on.
Lamp
(on)
Set Parameters
Setup and
Calibration
Set Delay UV to Frac
(x µl)
Set Parameters
6 Preparing the fraction collector
a) Check that the delay volume is correct. The delay
volume is equal to the volume between the UV ow
cell and the fraction collector (default 380 µl).
d) Check that the sensor is in the
correct position for the tube
size. The eluent tubing should be
positioned above the center of the
collection tube. Use the red sensor
control to position the tube holder.
b) Fill the fraction collector
with tubes*.
c) Adjust the height of the
delivery arm so that the
horizontal mark on the
tube sensor is at the same
level as the top of the
collection tubes.
L
oc
k kn
ob
5
m
m
Se
n
so
r
Co
n
t
r
ol
*See each cue card for information on the number of tubes to be
used.

11-0027-48 AC, 2007-09 • p3
e) Place the tubing holder over the length guide (small
hole) in the delivery arm, push the tubing down to the
bottom of the guide and tighten the nut. This ensures
that the correct length of the tubing is exposed.
Note: Make sure that the end of the tubing is cut
leaving a straight edge. For more information, see
ÄKTAprime plus User Manual .
Set Drop Sync Active
(yes)
Set Parameters
f) Re-install the tubing holder
into the delivery arm.
g) Rotate the rack by hand until the rear half of the tube
sensor rests against tube 1.
h) Press feed tube on the front panel. The bowl moves to
the correct position to collect the rst fraction in tube
1.
i) Make sure that drop synchronization is activated.
Note: Drop synchronization can only be used at ow
rates ≤ 3 ml/min.
7 Cleaning the sample loop and
connecting the column
a) Mount a sample loop between port 2 and port 6 that
is large enough to hold your sample.
b) Clean the loop and the injector ll port:
Inject (with a syringe) 5 times the loop volume of water
or binding buer through the injection ll port.
c) Connect the column between port 1 on the injection
valve (7-port valve) and the UV ow cell.
8 Filling the buer inlet tubing
Prepare the buers. Make sure that correct inlet tubing is
put into correct buer.
• If using an application template:
It is not necessary to perform the wash procedure
because this is included in the method.
• If using a method template:
Pre-ll all inlet tubings with buer. Select the
application template System Wash Method, select
the inlets to be washed and press OK.
The system is now prepared. Start the method.
9 Storage of the system
a) Clean the loop and the injector ll port:
• Inject (with a syringe) 5 times the loop volume of
water through the injection ll port.
• Repeat this step with 20% ethanol.
b) Flush the system ow paths:
• Put all used inlet tubings in water.
• Select the application template System Wash
Method, select the inlets to be washed and press
OK.
• For long-term storage of the system, repeat
step b) with 20% ethanol.
c) Shut down the system.
Tubing holder
HiTrap
TM
1/16" Male
UV monitor
1/16" Male
1/16" Male
HiPrep
TM

11-0027-48 AC, 2007-09 • p4
11-0027-48 AC, 2007-08 • p4
Buer exchange on HiTrap Desalting
1 Preparing the buers
•Use high purity water and chemicals.
• Filter all buers through a 0.45 µm lter before use.
Buer (port A1):
20 mM sodium phosphate, 0.15 M NaCl, pH 7.0*
Prepare at least 500 ml eluent.
* When performing buer exchange use the appropriate buer.
2 Preparing the sample
Pass the sample through a 0.45 µm lter.
The maximum recommended sample volume is 1.5 ml.
3 Preparing the system
a) Place the inlet tubing from port A1 (8-port valve)
and port B (2-port valve) in the buer.
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 ow cell (see Ordering
information on next page for suitable columns).
d) Fill the fraction collector rack with 18 mm tubes
(minimum 20) and position the white plate on the
fractionation arm against the rst 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 ll the loop.
Note: If the same sample is applied repeatedly, a
Superloop™ can be used. For information of how to
use it see the instructions for the 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 Controlled By:
prime should be displayed.
b) Use the arrow and OK buttons to move in the menu
tree until you nd Desalting HiTrap Desalting.
Theoretical gradient in Desalting HiTrap Desalting Application
Template.
c) Enter the sample volume and press OK to start the
template.
Desalting
HiTrap Desalting
Set Sample Inj. Vol
(00.0 ml) 00.0
Run Application Template
Press OK to start
Run data displayed
Application Template
Templates
100
50
6 3Min
Sample
Elution
Priming
& Equilibration
Total separation time = 9 min + sample application time
%B
0
0.05
0.10
0.15
AU
280nm
–– UV 280 nm
–– Conductivity
Inject
Histidine-tagged
protein
01
min
Sample: Histidine-tagged protein in
20 mM sodium phosphate,
0.5 M sodium chloride,
0.5 M imidazole, pH 7.4
Column: HiTrap Desalting 5 ml
Buffer (A1): 20 mM sodium phosphate,
0.15 M sodium chloride, pH 7.0
5 Typical result

11-0027-48 AC, 2007-09 • p5
Ordering information
Product Quantity Code No.
HiTrap Desalting 5 × 5 ml 17-1408-01
100 × 5 ml*11-0003-29
Superloop 10 ml 1 18-1113-83
* Pack size available by special order
Troubleshooting
High backpressure:
•Column clogged – Clean the column according
to instructions. Make sure the sample has been
centrifuged and/or ltered through a 0.45 µm lter.
•System clogged – Replace the column with a piece
of tubing. Check pressure. If backpressure > 0.3 MPa,
clean system according to manual.
Eluted sample still contaminated:
•Check that the sample load limit is not exceeded.

11-0027-48 AC, 2007-09 • p6
Buer exchange on HiPrep 26/10
Desalting
1 Preparing the buers
•Use high purity water and chemicals.
• Filter all buers through a 0.45 µm lter before use.
Buer (port A1):
20 mM sodium phosphate, 0.15 M NaCl, pH 7.0*
Prepare at least 500 ml eluent.
* When performing buer exchange use the appropriate buer.
Desalting
HiPrep Desalting
Set Sample Inj. Vol
(00.0 ml) 00.0
Run Application Template
Press OK to start
Run data displayed
Application Template
Templates
2 Preparing the sample
Pass the sample through a 0.45 µm lter.
The maximum recommended sample volume is 15 ml.
3 Preparing the system
a) Place the inlet tubing from port A1 (8-port valve)
and port B (2-port valve) in the buer.
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 ow cell (see Ordering
information on next page for suitable columns).
d) Fill the fraction collector rack with 18 mm tubes
(minimum 25) and position the white plate on the
fractionation arm against the rst 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 ll 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 Controlled By:
prime should be displayed.
b) Use the arrow and OK buttons to move in the menu
tree until you nd Desalting HiPrep Desalting.
Theoretical gradient in Desalting HiPrep Desalting Application
Template.
c) Enter the sample volume and press OK to start the
template.
100
50
13 5Min
Sample
Elution
Priming
& Equilibration
Total separation time = 18 min + sample application time
%B
0
0.1
0.2
0.3
0.4
0.5
0123 min
AU
280nm
–– UV 280 nm
–– Conductivity
BSA
Inject
Sample: BSA and sodium chloride
Column: HiPrep 26/10 Desalting, 53 ml
Buffer (A1): 20 mM sodium phosphate, 0.15 M NaCl, pH 7.0
5 Typical result

11-0027-48 AC, 2007-09 • p7
Ordering information
Product Quantity Code No.
HiPrep 26/10 Desalting 1 (53 ml) 17-5087-01
4 (53 ml) 17-5087-02
Superloop 10 ml 1 18-1113-83
Superloop 50 ml 1 18-1113-84
Troubleshooting
High backpressure:
• Column clogged – Clean the column according
to instructions. Make sure the sample has been
centrifuged and/or ltered through a 0.45 µm lter.
• System clogged – Replace the column with a piece
of tubing. Check pressure. If backpressure > 0.3 MPa,
clean system according to manual.
Eluted sample still contaminated:
• Check that the sample load limit is not exceeded.

11-0027-48 AC, 2007-09 • p8
e) Connect a sample loop large enough for your sample
between port 2 and 6 on the injection valve. Use a
syringe to manually ll 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 Controlled By:
prime should be displayed.
b) Use the arrow and OK buttons to move in the menu
tree until you nd Anity Purication any HiTrap.
Histidine-tagged protein purication, step
elution
1 Preparing the buers
•Use high purity water and chemicals.
• Filter all buers through a 0.45 µm lter before use.
Binding buer (port A1):
20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole,
pH 7.4 *
Elution buer (port B):
20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole,
pH 7.4
Prepare at least 500 ml of each eluent.
* Alternative binding buers:
5–40 mM imidazole can be included in the binding buer to
reduce unspecic binding of non-histidine-tagged proteins.
The concentration of imidazole is protein dependent and if the
protein of interest elutes or does not bind at a certain imidazole
concentration, then reduce the concentration.
2 Preparing the sample
a) Adjust the sample to composition of binding buer by:
• diluting the sample in binding buer or
• by buer exchange using HiTrap Desalting or HiPrep
26/10 Desalting.
b) Pass the sample through a 0.45 µm lter.
Note: If HisTrap FF crude column is used no ltration
nor clarication of the sample is needed.
3 Preparing the system
a) Place the inlet tubing from port A1 (8-port valve) in
the binding buer and the tubing from port B (2-port
valve) in the elution buer.
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 ow cell (see Ordering
information on next page for suitable columns).
d) Fill the fraction collector rack with 18 mm tubes** and
position the white plate on the fractionation arm
against the rst tube.
** The number of tubes to insert in the fraction collector varies with
the sample volume. Fill the fraction collector with 20 tubes + one
tube/ml sample. For example, if the sample volume is 10 ml, ll
the fraction collector with 20 + 10 = 30 tubes. However, note
that the maximum capacity of the fraction collector is 95 tubes,
limiting the sample volume to 75 ml.
Theoretical gradient in Anity Purication any HiTrap Application
Template.
Affinity Purification
any HiTrap
Set Sample Inj. Vol
(00.0 ml) 00.0
Run Application Template
Press OK to start
Run data displayed
Application Template
Templates
1 10 20 10 5 Min
100
50
Sample
Priming
Elution
Total separation time = 47 min + sample application time
Water wash &
priming
%B
Re-equili-
bration
Equilibration
c) Enter the sample volume and press OK to start the
template.
Note: If a 5 ml column is preferred, see cue card on
p.36.

11-0027-48 AC, 2007-09 • p9
Ordering information
Product Quantity Code No.
HisTrap HP 5 × 1 ml 17-5247-01
100 × 1 ml*17-5247-05
HisTrap FF 5 × 1 ml 17-5319-01
100 × 1 ml*17-5319-02
HisTrap FF crude 5 × 1 ml 11-0004-58
100 × 1 ml*11-0004-59
HiTrap Desalting 5 × 5 ml 17-1408-01
100 × 5 ml*11-0003-29
HiPrep 26/10 Desalting 1 (53 ml) 17-5087-01
4 (53 ml) 17-5087-02
Superloop 10 ml 1 18-1113-83
Superloop 50 ml 1 18-1113-84
Superloop 150 ml 1 18-1023-85
Troubleshooting
High backpressure:
•Column clogged – Clean the column according
to instructions. Make sure the sample has been
centrifuged and/or ltered through a 0.45 µm lter.
For unclaried samples using HisTrap FF crude, make
sure the sample has been lysed properly, e.g. using
thorough sonication and DNase treatment.
•System clogged – Replace the column with a piece
of tubing. Check pressure. If backpressure > 0.3 MPa,
clean system according to manual.
No binding:
•Check that the correct column is used.
• Check that the inlet tubing from each buer is
connected to the correct inlet port.
• Check that the composition and pH of the buers are
correct.
•Check that the sample has been adjusted to binding
buer conditions.
•Check that your sample contains target protein.
No elution:
• Check that the inlet tubing from each buer is
connected to the correct inlet port.
• Check that the composition and pH of the buers are
correct.
•Use alternative elution conditions according to the
column instructions.
•Check that your sample contains target protein.
5 Typical result
5 10 15 20 25 30 35 min
2.5
2.0
1.5
1.0
0.5
0
UV 280 nm
Programmed %B
Sample: Clarified homogenate of E. coli expressing histidine-tagged
protein
Column: HisTrap™ HP 1 ml
Binding buffer (port A1): 20 mM phosphate, 0.5 M NaCl, 20 mM
imidazole, pH 7.4
Elution buffer (port B): 20 mM phosphate, 0.5 M NaCl, 0.5 M
imidazole, pH 7.4
100
80
60
40
20
0
% B
AU
280 nm
*Pack size available by special order

11-0027-48 AC, 2007-09 • p10
c) Enter the sample volume and press OK to start the
template.
Note: If a 5 ml column is preferred see cue card on
p.36.
Histidine-tagged protein purication,
gradient elution
1 Preparing the buers
•Use high purity water and chemicals.
• Filter all buers through a 0.45 µm lter before use.
Binding buer (port A1):
20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole,
pH 7.4 *
Elution buer (port B):
20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole,
pH 7.4
Prepare at least 500 ml of each eluent.
* Alternative binding buers:
5–40 mM imidazole can be included in the binding buer to
reduce unspecic binding of non-histidine-tagged proteins.
The concentration of imidazole is protein dependent and if the
protein of interest elutes or does not bind at a certain imidazole
concentration, then reduce the concentration
2 Preparing the sample
a) Adjust the sample to composition of binding buer by:
• diluting the sample in binding buer or
• by buer exchange using HiTrap Desalting or HiPrep
26/10 Desalting.
b) Pass the sample through a 0.45 µm lter.
Note: If HisTrap FF crude column is used, nor ltration
nor clarication of the sample is needed.
3 Preparing the system
a) Place the inlet tubing from port A1 (8-port valve) in
the binding buer and the tubing from port B (2-port
valve) in the elution buer.
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 ow 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 rst 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 ll 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 Controlled By:
prime should be displayed.
b) Use the arrow and OK buttons to move in the menu
tree until you nd His Tag Purication HisTrap.
Theoretical gradient in His Tag Purication HisTrap Application
Template.
His Tag Purification
HisTrap
Set Sample Inj. Vol
(00.0 ml) 00.0
Run Application Template
Press OK to start
Run data displayed
Application Template
Templates
210 2020175Min
100
50
Sample
Priming
Wash
Elution
Total separation time = 74 min + sample application time
Buffer wash
%B
Re-equilibration
Equilibration