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Automated multi-step protein purification
System overview ...................
2
UNICORN overview ...........
4
ÄKTAxpress™ work flow:
1 Selecting protocols ........ 6
2 System preparations .... 10
3 Column preparations ... 12
4 Run preparations .......... 16
5 Starting a run ................. 18
6 Monitoring a run ........... 20
7 Evaluating the results ... 22
Affinity tag removal ............. 24
System cleaning .................... 25
Column cleaning .................. 26
Advanced zone ..................... 28
Flow charts ............................ 30
cue 18-1178-03 AA 2004-03 • p1
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Automated multi-step protein purification
Automated multi-step protein purification
System overview
System overview, continued
Control panel
Column block
Pause/Continue
1 – Affinity column 1
Tubing overview
RUN
2 – Affinity column 2
3 – Affinity column 3
or if using protocol E
– Desalting column
1
4 – Affinity column 4
or
– Ion exchange column
5 – Gel filtration
or
– Desalting column
or in a prepare run
– Affinity column 5
4
2
3
PAUSE
On/Standby
/Reset
Deep well
5
System no., status
and error codes
Next
breakpoint
Eject/Load
microplate
Sled for microplate
Deep well microplate,
96 square shaped wells
Manual injection
For details, see page 12.
Waste
Column valve
Column connection
3
Loop valve
Superloop connection
2
Outlets F3–F11
1
BYPASS
Column 4 BYPASS
connection
5
1
example
OUT 2 3 4
2
IN
4
5
5
1
4
Left holder
• Gel filtration column
• HiPrep Desalting column
3
1
3
2
Superloop
IN 4
5
OUT
Right holder
• Superloop™
• Pump rinsing solution
F3
F4
F5
F6
Flow through sample 1
sample 2
sample 3
sample 4
F7 Nonselected peaks,
F8
F9
F10
sample 1
sample 2
sample 3
sample 4
Buffers A1, A2
B1, B2
S1
S2
S3
S4
Sample inlets
Waste*
Sample 1
Sample 2
Sample 3
Sample 4
Buffers A3–A8
Grouping buffer containers
If the same buffer is used for several
systems, one buffer container can feed
several systems.
* Waste From injection valve port 2
(manual sample injection)
* Waste From injection valve port 3
(e.g. system wash)
For more information about buffer inlets,
see page 8.
F11 Metal ion waste
Recommended tubing positions
Waste
Detailed flow
charts
Frac
F3
A
H
Outlet Valve
F4
F11
F5
F10
See page 30–34.
F6
F9
F7
F8
Fraction Collector
OutV
0.2 MPa
Loop
L1
Restr
L3
L2
L4
L5
C3in
Cond
C2in
C4in
C5in
C3
C4
C2
L1
Bypass
BP
C1in
C5
Cond
C1
BP
L1
L5
L2
UV
L4
BP
Bypass
BP
L4
L5
L3
L3
L2
L1
UV
Loop Valve
C5out
C1out
C2out
C1
C4out
C3out
C2
Manual
inject
C4
C3
LoopV2
C5
Waste
ColV
Waste
Column Valve
Injection Valve
Safety
MixInj
P
A8
Inlet Valve
S4
A7
Read the ÄKTAxpress Safety Handbook
before using the system.
Press
A6
Mix1
S1
A3
NO
NC
A1
B2
IN
SW1
SW2
SW1
NC
Samples
NO
B1
A2
SW2
AirS
Pump
cue 18-1178-03 AA 2004-03 • p3
S2
A4
IN
cue 18-1178-03 AA 2004-03 • p2
S3
A5
Mix2
Mixer
Buffers
Pump
Air sensor
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Automated multi-step protein purification
Automated multi-step protein purification
UNICORN™ for ÄKTAxpress, overview
Run preparations – Method Editor
UNICORN for ÄKTAxpress, overview, continued
See 4 Run preparations,
page 16
Method Editor wizard
System Control wizard
The Method Editor wizard is used to create Method
Plans.
The System Control wizard is used to start runs based on
Method Plans. Example input information: Systems to
use, number of samples per system and sample
information.
Prepare
Purify
Maintain
Method plans
Starting a run – System Control
See 5 Starting a run,
page 18
Separation systems
Summary
check list
Default
parameters
View or modify
default parameters
Enter
Advanced
Zone
Save
Method Plan
Prints Summary
check list.
Sample
information
The run
starts with
Pause.
Evaluating the results – Evaluation
Run procedures
See 7 Evaluating the
results,
page 22
Result files
RUN
Advanced zone
PAUSE
Click Continue in UNICORN, or
Press Continue button on system.
RUN
Pooling protocols
Save
Method Plan
Reports
END
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Automated multi-step protein purification
Automated multi-step protein purification
1 Selecting protocols, columns and buffers
Selecting protocols, columns and buffers, continued
Protocols supported
Columns supported
Multi step protocols
Affinity Chromatography
AC
Desalting
Ion Exchange Chromatography
Single step protocols
AC
AC
AC
AC
DS
DS
IEX
DS
IEX
DS
IEX
DS
GF
DS
AC column selection
Volume [ml]
1 or 5
1 or 5
1 or 5
1 or 5
10
53
1
2
1
2
1 or 6
1 or 6
1
1
120
120
1 or 5 ml column
• Depends on required yield.
• If many purification steps are used, start with a 5 ml
column.
• For large sample volumes, use a 5 ml column
to save time.
• To increase protein purity, overload the column.
DS column selection
HiTrap or HiPrep
• Depends on loading volumes:
– Up to 3 ml: Use 2 × HiTrap Desalting.
– Up to 10 ml: Use HiPrep Desalting.
Gel Filtration or Desalting
GF
Protocol name
A
B
C
D
E
F
G
Protocol steps
2
2
3
4
4
1
1
Optional tag removal
•
•
•
•
•
Max no. of samples
4
4
3
3
2
4
4
•
•
•
•
•
•
•
•
•
•
• HiTrap Q HP and SP HP:
•
• RESOURCE Q and S:
15 µm
• Mono Q and S 5/50 GL:
10 µm
0.2
0.2
++
+
++
++
+++
Run time2), per sample [h]
– at room temperature
– in cold room
2.7
3.3
1.2
1.4
2.2
2.6
3.1
3.6
4.3
5.1
1)
2)
For ordering numbers see page 40.
For guidance on column selection, see also the
User Manual.
IEX column selection
Required resolution
The smaller bead the higher resolution:
Sample characteristics1)
– Buffer exchange
– Charge homogeneity
– Size homogeneity
– Purity increase
Run time2), total at max.
number of samples [h]
– at room temperature
– in cold room
GF
Column
HisTrap™ HP
HiTrap™ Chelating
GSTrap™ FF
GSTrap HP
HiTrap Desalting × 2
HiPrep™ 26/10 Desalting
HiTrap Q HP
HiTrap Q HP × 2
HiTrap SP HP
HiTrap SP HP × 2
RESOURCE™ Q
RESOURCE S
Mono Q™ 5/50 GL
Mono S™ 5/50 GL
HiLoad™ 16/60 Superdex 75 prep grade
HiLoad 16/60 Superdex 200 prep grade
•
+
The longer column the higher resolution:
1.6
2.0
• 2 columns in series give higher resolution.
Anion exchange. e.g. RESOURCE Q
10.4
12.8
4.3
5.2
6.3
7.4
9.3
10.7
8.6
10.1
0.6
0.7
• The pH of the buffer should be 1 pH unit above the
pI of the protein(s).
6.1
7.6
Cation exchange. e.g. RESOURCE S
Achieved after the last purification step.
Run times are approximate, sample loading time is not included.
Tip
• The pH of the buffer should be 1 pH unit below the
pI of the protein(s).
Tip
Use high purity liquids
Grouping samples
GF column selection
For best purification results, use high purity
deionized water and chemicals. Filtering of liquids
through a 0.45 µm filter and degassing the liquids is
recommended.
Considerations when running several separation
systems in parallel:
Separation size
To maximize the success rate, run similar proteins on
the same system, for example:
• Separation range:
Some examples of supported columns.
• To maximize GF performance, run small proteins
<70 kD on one system and large proteins >70 kD
on another system.
–
3–70 kDa: Use Superdex™ 75 prep grade.
– 10–600 kDa: Use Superdex 200 prep grade.
• To maximize IEX performance, run proteins with
low pI on one system and proteins with high pI on
another system.
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34 µm
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Automated multi-step protein purification
Automated multi-step protein purification
Selecting protocols, columns and buffers, continued
Buffers and solutions
DS buffer suggestions
If preparing for...
Buffer types needed
for AC: • binding buffer
• elution buffer
• cleavage buffer (optional) • extra wash buffer (optional)
for DS: • one buffer type per run (two for protocol D)
for IEX: • binding buffer
suggested buffer
binding using
HisTrap
binding using
HiTrap Chelating
extra wash2)
50 mM Tris-HCl pH 7.5,
0.5 M NaCl, 20–40 mM imidazole1)
50 mM Tris-HCl pH 7.5,
0.5 M NaCl, 5–40 mM imidazole1)
50 mM Tris-HCl pH 7.5, 0.5 M NaCl,
100 mM imidazole
50 mM Tris-HCl pH 7.5,
0.5 M NaCl, 50 mM imidazole
50 mM Tris-HCl pH 7.5, 0.5 M NaCl,
500 mM imidazole
AC buffer suggestions for GST-tagged proteins
If performing...
suggested buffer
include e.g. 10% glycerol in a suitable
buffer (e.g. 50 mM Tris-HCl, pH 7.5,
150 mM NaCl)
binding to AIEX
suggested buffer
(depends on the pI of the protein)
50 mM Tris-HCl pH 8.0
binding to CIEX
20 mM MES pH 6.0
elution from AIEX
50 mM Tris-HCl pH 8.0, 1 M NaCl
elution from CIEX
20 mM MES pH 6.0, 1 M NaCl
GF buffer suggestions
If preparing for...
suggested buffer
further studies
a suitable buffer, e.g.
50 mM Tris-HCl pH 7.5, 150 mM NaCl
include e.g. 10% glycerol in a suitable
buffer (e.g. 50 mM Tris-HCl pH 7.5,
150 mM NaCl)
protein storage
The imidazole concentration is protein dependent.
x% of extra wash buffer is added to the binding buffer. The x%
should be specified in the Method Wizard. See User Manual for
details.
3) In the affinity tag removal protocols, the affinity columns can be
equilibrated with either AC-binding/wash buffer or an alternative
cleavage buffer prior to protease injection.
1)
2)
binding using
50 mM Tris-HCl pH 7.5,
GSTrap HP or FF
150 mM NaCl, 1 mM DTT
cleavage using
50 mM Tris-HCl pH 7.5,
PreScission™ protease 150 mM NaCl, 1 mM EDTA,
on GSTrap HP or FF3) 1 mM DTT
step elution
protein storage
If for example...
If performing...
step elution
20 mM MES pH 6.0
IEX buffer suggestions
AC buffer suggestions for His-tagged proteins
cleavage using TEV
on HisTrap HP or
HiTrap Chelating HP3)
50 mM Tris-HCl pH 8.0
CIEX
• elution buffer
for GF: • one buffer type per run
suggested buffer
AIEX
50 mM Tris-HCl,
10 mM reduced gluthathione, pH 8
Inlet buffers and solutions
Inlet
Purification run
and column preparations
Stripping, charging (AC)
and blank run
A1
Affinity binding buffer (A)
Affinity binding buffer (A)
GST A buffer
B1
Affinity wash buffer (B)
(additional wash)
–
1 M NaCl
A2
Ion exchange binding
buffer (A) / Desalting buffer
–
B2
Ion exchange elution buffer (B)
–
2 M NaCl
A3
Affinity elution buffer (B)
Affinity elution buffer (B)
30% isopropanol
A4
Gel filtration buffer or
Desalting buffer (final step)
–
0.2 M NaOH
A5
–
Deionized water
Deionized water (cleaning)
A6
0.5 M NaOH (if NaOH wash
between samples is chosen)
–
0.5 M NaOH
A7
Cleavage buffer
(tag removal only)
Metal ion charging solution
(e.g. 0.1 M NiSO4)
1.0 M NaOH
A8
–
Metal ion stripping solution (e.g.
His-Affinity A buffer with 50 mM EDTA)
6 M Guanidine HCl or
1% TritonX100
cue 18-1178-03 AA 2004-03 • p8
CIP – Cleaning in place
Ion exchange binding buffer (A)
cue 18-1178-03 AA 2004-03 • p9
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Automated multi-step protein purification
Automated multi-step protein purification
2 System preparations
System preparations, continued
Automatic filling of the inlet tubing (Prepare)
Manual filling of the inlet tubing
Other purging procedures
Purging the pump with methanol
General
Before starting a run, all inlet tubing must be filled to
remove air bubbles.
Purge the pump and system
A small pump flow of 0.1 ml/min may be used. Set the
flow in System Control : Manual : Pump.
Air remaining in the system may be removed by purging
the pump and system by manually selecting Pump Wash
and System Wash.
Both pump heads must be purged with methanol if:
– The system has been left unused for a week, or longer,
or
– The pump has been run dry.
System preparations can be made:
– As individual Prepare methods, as described here, or
– As part of the purification run, see page 16.
If the inlet tubing is without air bubbles an automatic
Prepare method can be used to fill the buffer and sample
inlet tubing with buffer/solution. If there is air in the
tubing, perform a Manual filling of the inlet tubing.
Filling inlet tubing A1–A8 and S1–S4
• Immerse tubing in the liquid containers. Put the
sample inlet tubing A1 into buffer A.
Note: When performing a Prepare, Purification or
Maintain run, an initial system wash will be
included in the method.
• Connect a syringe
to the purge valve.
• Immerse A1 tubing in the buffer to be used.
Creating the method plan
• Select System Control : Manual : PumpWash
or System Control : Manual : SystemWash.
• Click the Method Wizard icon in the
Method Editor module.
• Set the injection valve to position Waste:
– Select System Control : Manual : Flow path :
InjectionValve : Waste and click Execute.
• Turn the purge
valve to open it.
• On the last page, click Finish
(or to enter the Advanced zone, click Next).
• Run the pump at 20 ml/min for 2 minutes:
– In System Control : Manual : Pump, set a flow
rate of 20 ml/min.
– Click Execute to start the flow and run the pump for
2 minutes.
– Stop the pump by clicking Pause.
• In the Save As page, type the name of the method
plan and click OK.
Performing the run
• Carefully move the inlet tubing A1 to the flask
containing about 200 ml of methanol.
• Run the pump at 50 ml/min for 2 minutes:
– Set the flow rate to 50 ml/min and click Execute.
– Click Continue to start the flow and run the pump for
2 minutes.
– Stop the pump by clicking Pause.
• Slowly draw solution
into the syringe.
When fluid starts to
enter the syringe,
continue to draw a
few milliliters before
closing the purge
valve. Check that
there is no visible air
left in the tubing.
• Run the pump at 20 ml/min for 1 minute:
– Set the flow rate to 20 ml/min and click Execute.
– Click Continue to start the flow and run the pump for
1 minute.
– Stop the pump by clicking Pause.
• Carefully move the inlet tubing A1 back to the flask
containing water.
• Repeat the procedure for A2–A8 and S1–S4.
Set the inlet valve to the appropriate positions in
System Control : Manual : Flowpath.
• Repeat the procedure for B1 and B2.
– To fill B1 set valve positions:
In System Control : Manual : Pump : Gradient
select Target 100%B and Mode A1/B1.
When the inlet is filled, reset Target %B to 0%.
– To fill B2 set valve positions:
In System Control : Manual : Pump : Gradient
select Target 100%B and Mode A2/B2.
When the inlet is filled, reset Target %B to 0%.
cue 18-1178-03 AA 2004-03 • p10
• Prepare two flasks:
– 200 ml of methanol
– 300 ml of deionized water
• Put both Waste tubings from the injection valve into
a waste flask.
• Select Prepare : System (for column preparation see
page 12).
• Run the Prepare method plan as any
other run using Instant Run. See page
18.
Note: All inlet tubing that will be used in the next
purification run should be filled manually before
purging the pump.
• Carefully immerse the inlet tubing A1 in the flask
containing at least 300 ml of deionized water.
• Select New or open an existing method plan to edit.
• Select Fill buffer inlets and/or Fill Sample Inlets and
specify inlets.
The purging will maintain the pumping capacity and
protect the pump piston seals.
Tip
To run the pump for a specific length of time, the
instructions PauseTimer and EndTimer can be used.
The instructions are found in:
System Control : Manual : Other.
cue 18-1178-03 AA 2004-03 • p11
• Run the pump at 10 ml/min for 10 minutes:
– Set the flow rate to 10 ml/min and click Execute.
– Click Continue to start the flow and run the pump for
10 minutes.
– Stop the pump by clicking End.
• Carefully move the inlet tubing A1 to the flask
containing the buffer that will be used during the
next purification run.
Note: Remember to fill inlet tubing A1 with the buffer to
be used, as described on page 10.
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Automated multi-step protein purification
Automated multi-step protein purification
3 Column preparations
Tip
Check for leakage during column equilibration.
• Run the Prepare method plan as any
other run using Instant Run. See page
18.
Note: Check that appropriate buffers/solutions have
been prepared and placed at the correct inlets.
See tables page 8.
1 Creating the method plan
Connecting columns
• Click the Method Wizard icon in the
Method Editor module.
Drop-to-drop
• Select New or open an existing method plan to edit.
• Select Prepare : Columns and select column
preparation options as required.
GF
AC
IEX
1/16" Female/
M6 Male
To avoid air bubbles, use the
drop-to-drop procedure when
connecting columns:
HiTrap 5 ml
Column preparations can be made:
– As individual Prepare/Maintain methods,
as described here, or
– As part of the purification run, see page 16.
Connecting tubing to columns
2 Performing the run
HiTrap 1 ml
Column preparations
Column preparations, continued
1/16" Female/
M6 Female
1/16" Male/
M6 Female
and
GSTrap,
HisTrap
2 Set the column valve to
the required position.
Union M6 male to
1/16" female
18-1112-57
Union M6 female to
1/16" male
18-1112-58
Union M6 female to
1/16" female (PEEK)
18-1123-94
Union 1/16" male to
1/16" male
18-1120-93
On-line filter
18-1112-44
DS
On-line
filter
1/16" Male/
M6 Female
1 Start the pump with a low
flow rate.
Ordering numbers:
1/16" Female/
M6 Male
Column block
Column positioning overview
AC(1) AC(2) AC(3) AC(4)
GF
B
4
AC(1) AC(2) AC(3) AC(4)
DS
C
3
AC(1) AC(2) AC(3)
IEX
DS
D
3
AC(1) AC(2) AC(3)
IEX
DS
E
2
AC(1) AC(2)
DS
IEX
GF
F
4
–
–
–
–
DS
G
4
–
–
–
–
GF
RESOURCE 6 ml
4
RESOURCE 1 ml
A
Mono Q 5/50 GL
Protocol No. of Column type (sample) in column position
samples
1
2
3
4
5
HiPrep 26/10 Desalting
IEX
HiLoad 16/60 Superdex
3 Fix the tubing to the column
drop-to-drop.
1/16" Female/
M6 Male
1/16" Male/
1/16" Male
1/16" Female/
M6 Female
Column block
Tip
• On the last page, click Finish
(or to enter the Advanced zone, click Next).
Replace the union’s internal capillary with a
1 mm peek tubing (brown) to reduce backpressure,
if required.
• Finally, in the Save As page, type the name of the
method plan and click OK.
View film in On-line Cue Card
cue 18-1178-03 AA 2004-03 • p12
Using ÄKTAxpress
cue 18-1178-03 AA 2004-03 • p13
350 mm tubing1)
1)
The standard system tubing should be replaced by the
350 mm capillary tubing available in the accessory kit.
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Automated multi-step protein purification
Automated multi-step protein purification
Column preparations, continued
For specification of buffers and inlets, see page 8.
Column equilibration
AC
DS
IEX
Customized equilibration AC
GF
Prepare/Column equilibration will equilibrate columns
with buffer before starting a purification run.
All four column types can be selected in the same run.
Column
Inlet
Volume Flow [ml/min]
RT
CR
HisTrap HP, HiTrap Chelating
(1 ml / 5 ml)
GSTrap HP/FF (1 ml / 5 ml)
HiTrap Desalting
HiPrep 26/10 Desalting
HiTrap Q/SP HP
RESOURCE Q/S (1 ml / 6 ml)
Mono Q/S
HiLoad Superdex 75/200 pg
A1
A1
A2/A4
A2/A4
A2
A2
A2
A4
5 CV
5 CV
5 CV
5 CV
5 CV
5 CV
5 CV
2 CV
1/10
1/10
10
10
1
4/6
1
1.5
IEX
GF
With custom equilibration up to 5 columns of each type
may be selected and up to 9 different solutions may be
used.
0.8/8
0.8/8
8
8
0.8
3.2/4.8
0.8
1.2
AC Columns – Metal ion charging
DS
Customized equilibration is a procedure that equilibrates
the column with an optional number of solutions. Only
one column type can be included. The procedure is
used, for example, to remove ethanol from new columns,
using deionized water before equilibration with buffer.
Customized equilibration is a Maintain method plan:
• Select Maintain.
• Select Column CIP (Rigorous) / Customized
Equilibration
• Make appropriate selections on each page.
Note: The first column should be positioned in column
position 1, the second column in position 2, etc.
AC Columns – Affinity blank run
AC
AC
Purpose
Purpose
To charge new or stripped HiTrap Chelating HP, or
stripped HisTrap HP columns with metal ions (e.g. Ni2+).
Several columns can be prepared simultaneously.
It is recommended to run a blank run:
• Prior to a first time usage of an affinity column, or
• After metal ion charging.
Manual preparations
IEX columns – Ion exchange blank run
Note: Put the outlet tubing F11 in a liquid container for
collection of any metal waste, if required.
Purpose
IEX
To provide ion-exchange columns with exchangable
counter ions. A blank run should be performed before
first-time usage and after long-term storage.
Metal ion charging (1 ml / 5 ml)
Step 1
Solution
Inlet Volume
Deionized A5
water
5 CV
Flow [ml/min]
RT
CR
1/5 0.8/4
Step 2
Solution
Inlet
Metal ion A7
solution
Volume Flow [ml/min]
RT
CR
1 CV
1/5
0.8/4
Step 3
Solution
Inlet
Deionized A5
water
Volume Flow [ml/min]
RT
CR
5 CV
1/5 0.8/4
Affinity blank run (1 ml / 5 ml)
Step 1
Buffer A
A1
5 CV
1/5
0.8/4
Step 2
Buffer B
A3
5 CV
Step 2
Buffer B
B2
5 CV
1/5
0.8/4
Step 3
Buffer A
A1
10 CV
Step 3
Buffer A
A2
10 CV
1/5
0.8/4
1
4/6
1
0.8
3.2/4
0.8
Ion exchange blank run
Step 1
Buffer A
A2
5 CV
– HiTrap Q/SP HP
1
– RESOURCE Q/S (1/6 ml) 4/6
– Mono Q/S
1
0.8
3.2/4.8
0.8
cue 18-1178-03 AA 2004-03 • p14
1
4/6
1
0.8
3.2/4
0.8
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Automated multi-step protein purification
Automated multi-step protein purification
4 Run preparations
Run preparations, continued
Creating a method plan for the protein
purification
Use the Method Editor wizard to create a new method
plan or to change an existing method plan.
• Select Purify.
Advanced zone
General Advanced Settings
• Select procedures to be included in the run:
– Include System Preparation
see page 10
– Include Column Preparation
see page 12
General
Some options can be set in the Advanced zone page
General Advanced Settings.
• Select Purification Protocol and Include Tag Removal
(see page 24) if required.
Creating the method plan
• Make appropriate selections on each page.
• Click the Method Wizard icon in the
Method Editor module.
• On the last page, click Finish
(or to enter the Advanced zone, click Next).
• Select New or open an existing method plan to edit.
• Finally, in the Save As page, type the name of the
method plan and click OK.
Note: Do not change default parameter values in a
method plan unless the result is clearly
understood
understood. Changing one parameter value
might require adjustments of other parameter
values as well.
Main
Prepare
Purify
Maintain
Purification protocol
A:
B:
C:
D:
E:
F:
G:
Include
System
Preparation
Column
Equilibration
In the Advanced zone,
all main parameter
Advanced zone
values can be viewed,
and modified if required.
Examples of parameters that may
be modified:
• Wash volumes
• Flow rates
• Gradient slopes
• Watch parameters for peak detection
AC–GF
AC–DS
AC–DS–IEX
AC–DS–IEX–DS
AC–DS–IEX–GF
DS
GF
Getting help
To get help about
parameter setting,
click Help in the
wizard boxes.
Setup Result file folders
Select Store Results in a Main Folder and enter the name
of a new or existing folder in Main Folder Name.
Specify path to import file
If an import file is going to be used for specification of
sample information, the file path can be set in
Import File Location.
Tip
If the import files are saved in the default folder
MethodWizardImport, there is no need to specify the
path.
Disabling air sensor aided loading
Air sensor aided loading of samples is enabled as
default. To disable air sensor loading uncheck
Enable AirSensor Controlled Sample Loading.
Tag Cleavage
Running Condition
Prepare Column
Last Page
Fill
Affinity
...
...
Desalting
Ion Exchange
More information about Advanced zone
Room Temp.
Cold Room
Prepare System
See page 28.
Additional parameters
...
...
Tip
Column selection
Use the Note field in the Save As
page to specify changes made to
the default settings.
Assisted...
...
...
Gel filtration
Method plan
Save As
Finish
OK
Method plan
Save As
Next >
Advanced zone
Finish
OK
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Creating a Method Plan
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Automated multi-step protein purification
Automated multi-step protein purification
5 Starting a run
Tip
Starting a run, continued
Select run parameters
Final preparations
3 Select method plan
8 Check the flow path
• In UNICORN System Control, click
Instant Run.
Buffer and sample tubing inspection
• If Fill sample inlets is included in the method plan,
the inlets should be placed in affinity A buffer.
During the run, the system will pause after initial
buffer filling, and a message will appear requesting
each sample inlet tubing to be moved to its sample.
Buffer volumes required
It is possible to print out a Summary check list to get
a list of the buffer volumes and inlet positions for the
run before preparing the system and columns.
To do this:
1 Start with step 3 and continue to 7.
2 Print out the Summary check list and instead of
starting the run, press Cancel.
• Select the required Method plan
from the list.
4 Select systems for the run
• Select on which System(s) the
method plan will be run and the
Number of Samples on each
system.
5 Specify samples
• Check that the tubing is fully immersed in the
containers. Fix the tubing, for example, by:
– Fitting the inlet filter weights on the tubing, or
– Running the tubing through the rubber cap
included in the accessory kit.
• If Fill sample inlets is not included, gently move the
sample inlet tubing to each sample.
• Immerse the waste and outlet tubing in appropriate
containers according to the Summary check list.
• Secure the tubing with the tubing holder.
• Make sure:
– there is enough buffer available
– the correct inlet is placed in each buffer
– the Superloop is attached, if to be used.
Start the run
Air bubbles inspection
• Click Run to start the run on the
selected systems. The systems will
immediately enter Pause.
• Inspect the inlet tubing for air bubbles.
All tubing up to the pump heads should be inspected.
Make sure that no air enters the tubing. Place the
tubing close to the bottom of the liquid container but
not too tight against the bottom.
11 Start the run
RUN
PAUSE
12 Final check
S1 S2 S3 S4
• Peform a final check that tubing, columns and solutions are placed according to the Summary check list.
Tip
Prepare system and columns
1 Prepare the buffer, inlets, outlets and waste
• Prepare buffers required for the run.
• Immerse or connect all inlet tubing to the appropriate
liquid containers as described on page 8 and make
sure the system is prepared as described in System
preparations page 10.
• Immerse unused tubing in e.g. 20% ethanol to avoid
air entering the tubing.
• If Fill sample inlets is not included in the method plan
to use, make sure the sample inlet tubing is filled
with affinity buffer A, see System preparations page
10.
2 Connect columns and superloop
• Enter identification names
for the samples, either via
the keyboard or using a
bar code reader.
• For each sample, enter:
– isoelectric point, pI,
– extinction coefficient for the protein, Ext Coeff,
– molecular weight of the protein, MW.
The data is automatically imported from an import file,
if it has been prepared and placed in the specified
folder.
• Enter optional text, e.g. culture batch number.
cue 18-1178-03 AA 2004-03 • p18
Buffers
A1, A2, B1, B2
Buffers A3–A8
Samples
S1–S4
9 Prepare the microplate
• Press the Eject button on the system to
eject the fraction collector sled, if required.
6 Edit result file location and names
• Place an empty deep well microplate on the
sled and check that the
labelling H and A match
the labelling on the system.
• If required, edit the folder path and file names of the
result files to be created.
• Press the Eject button
again to load the microplate.
7 Print Summary check list
• Print out the Summary check list by clicking Print.
• Connect selected columns as described on page 12
and prepare them according to the Column
preparations description.
• Connect the Superloop if it is to be used, see Affinity
tag removal, page 24.
Sensitive samples can be attached to the system at
this point.
10 Prepare the samples
• Prepare the samples and clarify them using
centrifugation and/or filtration through a 0.45 µm filter.
• Place the sample tubes in the tube or flask holder.
View film in On-line Cue Card
Initiating Runs
13 Continue the run
• Click Continue on one system at a
time or Continue all to continue
the run on all systems. Alternatively, press the Continue button on
each individual system.
• If Tag Removal was selected, a wizard will give
guidance on how to fill the Superloop manually. See
Affinity tag removal page 24.
Single step protocols (F and G)
• For single step protocols, a wizard will give guidance
on how to fill the loops with sample(s) manually.
Tip
For single step protocols, manually fill the loop to be
used with the running buffer, before starting the
protocol.
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RUN
Note: If the run starts with a system
wash, the system will enter pause again.
Therefore, do not press Continue more than once.
System Check and Start
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Automated multi-step protein purification
Automated multi-step protein purification
6 Monitoring a run
Monitoring a run, continued
Monitoring a run is performed in System Control.
Run data
Right-click the view pane
and select Properties to
select run data to be
displayed.
View single or all systems
The run data of all
systems can be
viewed
simultaneously by
clicking Expand >>.
Return to single
system view by
clicking << Collapse.
Example of a 4-step purification run
Conductivity
UV
Empty loops,
discarded
Re-equilibration
of IEX
Wash 2
AC
DS
IEX
Re-generation
of columns
GF
Post-run
procedures
Panes
Click Panes to
select panes to
display.
wash
salt
fractionation
Sample loading
S1 S2 S3 S4
Zoom
Use the mouse dragand-drop box to
zoom in and out the
curve. Right-click to
reset zoom.
Run status
Each system’s status indicator shows the run status:
Status
Color
Example of events
Log book
Right-click to select
log book properties
to be displayed.
End .................. White
Run or Manual
Green
Hold ................ Yellow ............... Sample loading
Curves
Right-click to select
which curves to be
displayed.
Flow through
F3–F6
Nonselected
peaks F7–F10
Microplate
Run finished
When a system has entered End status the result can be
evaluated, see Evaluating the results page 22.
Tip
If there was a problem
See chapter Troubleshooting in the User Manual.
Cleaning after a run
After the run, if required:
• Empty drip plate
• Clean system
• Clean columns
Pause .............. Red .................. Pump-wash
Error ................ Yellow, flashing .. Air detected (e.g. running out of buffer)
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Monitoring a Run
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Automated multi-step protein purification
Automated multi-step protein purification
7 Evaluating the results
1 Find and open the result files
• Use the Recent Runs or the Find tab, in the Evaluation
module to locate the result file.
Note: Automatic pooling is not performed if the result
file is opened via the Files tab.
• Click
to expand the list for the result file. A result
file consists of:
– Sample loading chromatogram (11 below)
– Purification chromatograms for each sample
• Double-click a sample chromatogram to open it.
• If necessary, adjust the theoretical suggested pooling.
If the extinction coefficient has been entered in the
wizard, concentration and amount are automatically
calculated. Otherwise: enter the extinction coefficient
manually by marking a pool and then typing the value
in the extinction coefficient field. The concentration is
given in mg/ml, or M, and amount is given in mg, or
mmole, depending on the coefficient used.
The linearity of the UV monitor is limited. For more
information, see Technical specification in the User
Manual.
Evaluating the results, continued
Getting help
4 Print the Report
On-line Manuals and Cue Cards
• Select File : Report or click the Report icon.
• Select Xpress1sample, Xpress2samples,
Xpress3samples or Xpress4samples report format,
depending on how many samples are included in the
result file.
• Open the Help menu and select
Manuals to access the
on-line instructions:
– UNICORN User Manual
– ÄKTAxpress User Manual
– Cue Cards with films.
• The report format XpressActive Chrom can also be
selected if only the active zoomed window should be
included in the report.
• Click the Add to Pooling Protocol button to add the
adjusted pools to the Pooling protocol.
• Repeat the procedure for other chromatograms from
the same, or other, result files.
• Click the View Pooling Protocol button.
3 Print or save the Pooling protocol
The Pooling protocol can be used as a help when
making the physical pooling of the purified samples from
the microplate.
2 Adjust pooling and add to Pooling protocol
The chromatogram is displayed and UNICORN will
automatically display a suggested theoretical pooling of
the fractions. The pooled fractions are listed in a table
below the chromatogram and the pooled peaks are
numbered sequentially in the chromatogram.
• To print the Pooling protocol:
Click the Print button to print the protocol on the
default printer.
• Click Edit or Preview to inspect and modify the report
format if needed.
• Click the Print button.
• Choose which pages and how many copies to print
and click OK.
• To save the Pooling protocol as a file:
Click Export and save the protocol in one of the
following formats: text (.txt), Excel (.xls), HTML (.htm).
Context-specific Help
• Click the Help button in the dialog box,
or press F1.
Example of Method Editor wizard help:
Printed instructions
Printed information for ÄKTAxpress:
Only adjacent fractions will be pooled. The fraction
numbers for each pool are listed in the table as a range
in retention order, e.g. A6–A7 etc.
Book
PDF
On-line
User Manual
•
•
•
Safety Handbook
•
•
–
If the pooling suggestion is not performed, choose
Operations : Pool.
Installation Guide
•
•
–
UNICORN manuals
•
•
•
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Evaluating the Results
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For ordering numbers see page 40.
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Automated multi-step protein purification
Automated multi-step protein purification
System cleaning
Affinity tag removal
General
Filling the Superloop
Affinity tag removal can be performed in all multi-step
protocols.
The Superloop can be filled:
– With assistance from a Purify method plan.
– With assistance from a Prepare method plan,
if Assisted Manual Loading of Superloop has been
selected.
– Manually. There is a higher risk of introducing air into
the system with this procedure.
Creating the method plan
• In the Purify or Assisted Loading method plan, select
Tag Cleavage to add an affinity tag removal step to the
method plan.
Assisted filling
• Choose Incubation time and select Fill Columns with
Cleavage buffer if the ordinary binding buffer should
not be used.
During Assisted Manual Loading
of Superloop, dialog boxes will
give guidance to filling the
Superloop a the beginning of the
run. The filling is performed via a
syringe connected to the injection
valve.
• Make appropriate selections on each page.
• On the last page, click Finish.
• In the Save As page, type the name of the method
plan and click OK.
• During the run, follow the
dialog boxes and press
Continue between each step.
1 Fill syringe with buffer and
insert syringe in valve.
2 Inject buffer.
3 Fill syringe with protease
solution and insert syringe in valve.
4 Inject protease solution.
Calculate protease amount
• Calculate the volume and concentration of protease
needed. Use, for example, the Excel file available on
the Installation CD.
Clean system
Leaving the system for some days
Purpose
If the system is not going to be used for some days:
Use a bacteriostatic solution, for example deionized
water and 20% ethanol, as the two final wash solutions.
To wash all used tubing, including the loops, within the
system: up to 5 cleaning solutions can be used, one at a
time.
Creating the method plan
Superloop off-line cleaning
• Click the Method Wizard icon in the
Method Editor module.
See the Superloop instructions for guidelines on how to
clean the Superloop off-line.
• Select New or open an existing method plan to edit.
Note: The Superloop can not be cleaned automatically.
When starting a Loop Wash on position 5 make
sure to disconnect the Superloop and replace it
with a capillary loop.
• Select Maintain.
• Select Clean System.
• Select the parts to be cleaned.
• On the last page, click Finish
(or to enter the Advanced zone, click Next).
• In the Save As page, type the name of the method
plan and click OK.
Manual preparation
• Prepare a sufficient volume of the wash solutions
(max. 5).
• Wash the outside of the inlet tubing with deionized
water and/or ethanol.
• Immerse the tubing in a container of cleaning solution.
• If the column valve is to be cleaned:
Remove the columns and reconnect the tubing to use
as bypass column connections.
• Check that loop 5 is a capillary loop if the loop valve is
to be cleaned.
Connect the Superloop
Performing a Clean System run
• Fit the Superloop in the right
hand holder.
• Run the Clean System run as any other
run using Instant Run. See page 18.
Top
Note: Make sure the Superloop
is not hindering the
ejection of the microplate.
• A message will prompt when to manually change
cleaning solutions. Inlet A1 is used for cleaning
outlets, loops and column valve.
• Connect the Superloop to the
Loop Valve:
– Top to OUT 5
– Bottom to IN 5
2
3
Superloop
5
1
5
4 3
2
The protease can be injected directly into the Superloop
before starting the run.
IN
4
1
See page 32 for the flow chart of system cleaning.
Manual filling off-line
• Connect a syringe to the lower port of the Superloop
and fill it. Avoid introducing air bubbles.
0 ml
OUT
Manual filling in-line
See the User Manual.
Bottom
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Automated multi-step protein purification
Automated multi-step protein purification
Column cleaning
Column cleaning, continued
Note: Make sure all inlet tubing is filled before start.
See System preparations, page 10.
AC
Column CIP (Cleaning-In-Place)
Metal Ion Stripping (chelating columns)
Purpose
Purpose
To clean contaminated columns. For column cleaning
procedures and column storage instructions, please refer
to the instructions supplied with each column or to the
Amersham Biosciences web page:
www.chromatography.amershambiosciences.com
To remove metal ions before regenerating the HiTrap
chelating and HisTrap affinity columns. To recharge the
column, follow the instructions in Column preparations
page 12.
Creating the method plan
• Click the Method Wizard icon in the
Method Editor module.
• Select New or open an existing method plan to edit.
Note: Always remove metal ions before or directly after
storing the columns in ethanol.
Creating the method plan
• Select Maintain.
• Select New or open an existing method plan to edit.
• Select Maintain.
• Select Strip Affinity Columns.
• Select the type of column, number of columns, and
running condition.
• On the last page, click Finish
(or to enter the Advanced zone, click Next).
Rigorous can have up to 9 steps and the default
cleaning parameters can be changed.
• In the Save As page, type the name of the method
plan and click OK.
See the tables for specifications of the CIP procedures.
Performing a Metal Ion Stripping run
• On each page, select the parameter values.
• On the last page, click Finish
(or to enter the Advanced zone, click Next).
• Run the stripping run as any other run
using Instant Run. See page 18.
• In the Save As page, type the name of the method
plan and click OK.
Performing a CIP run
• Run the CIP run as any other run using
Instant Run. See page 18.
Step 1
Column1)
Solution
HisTrap HP, HiTrap Chelating
(1 ml / 5 ml)
1M NaOH
GSTrap HP/FF (1 ml / 5 ml)
6M GuaHCl or
1% Triton X-100
HiTrap Desalting
0.2M NaOH
HiPrep 26/10 Desalting
0.2M NaOH
HiTrap Q/SP HP
2M NaCl
RESOURCE Q/S (1 ml / 6 ml) 2M NaCl
Mono Q/S
2M NaCl
HiLoad Superdex 75/200 pg 0.5M NaOH
1)
• Click the Method Wizard icon in the
Method Editor module.
• Select cleaning type:
– Column CIP (Regular)
– Column CIP (Rigorous)
Regular is a 2-step cleaning where the cleaning
procedure is automatically adjusted for the specified
column to clean. The solutions and inlets are specified
on the Summary check list when starting the run.
Inlet
Flow [ml/min]
Volume RT
CR
Step 2
Solution
A7
A8
1 CV2)
2 CV
1/5
1/5
0.8/4
0.8/4
A4
A4
B2
B2
B2
A6
2 CV
2 CV
2 CV
3 CV
2 CV
1 CV
10
10
0.35
4/6
2
0.5
8
8
0.35
3.2/4.8
1.6
0.3
The columns must be filled with deionized water prior to CIP.
2)
IEX
GF
Strip of HisTrap and HiTrap Chelating (1 ml / 5 ml)
Procedure
Strip
Step 1
Solution
Inlet
His affinity
A8
A-Buffer with
50mM EDTA
Step 2
Deionized water A5
Volume
5 CV
Flow [ml/min]
RT
CR
1/5
0.8/4
5–10 CV
1/5
Volume
Flow [ml/min]
RT
CR
Deionized water A5
GST A-buffer
A1
10 CV
5 CV
1/5
1/5
0.8/4
0.8/4
Deionized water
Deionized water
IEX A-buffer
IEX A-buffer
IEX A-buffer
Deionized water
A5
A5
A2
A2
A2
A5
5 CV
5 CV
5 CV
5 CV
10 CV
2 CV
10
10
1
4/6
2
0.5
8
8
0.8
3.2/4.8
1.6
0.3
Step 2
Solution
Inlet
Flow [ml/min]
Volume RT
CR
Inlet
Pauses 1 hour.
CIP Rigorous, recommended solutions and inlets
Column
HisTrap HP, HiTrap Chelating
(1 ml / 5 ml)
GSTrap HP/FF (1 ml / 5 ml)
HiTrap Desalting
Step 11)
Solution
1M NaOH
6M GuaHCl
1mg pepsin/ml in
0.1M acetic acid,
0.5M NaCl
HiPrep 26/10 Desalting
1mg pepsin/ml in
0.1M acetic acid,
0.5M NaCl
HiTrap Q/SP HP
2M NaCl
RESOURCE Q/S (1 ml / 6 ml) 1M NaCl
Mono Q/S
1M NaCl
HiLoad Superdex 75/200 pg 1M NaOH
Inlet
Flow [ml/min]
Volume RT
CR
A7
1 CV2)
A8
2 CV
A7 or 1 CV3)
A8
1/5
1/5
5
0.8/4
0.8/4
4
Deionized water A5
GST A-buffer
A1
0.2M NaOH
A4
10 CV
5 CV
2 CV
1/5
1/5
5
0.8/4
0.8/4
4
A7 or 1 CV3)
A8
10
8
0.2M NaOH
A4
2 CV
10
8
B2
B1
B1
A7
0.25
4/6
0.5
0.5
0.20
3.2/4.8
0.4
0.4
Deionized water
Deionized water
Deionized water
Deionized water
A5
A5
A5
A5
2 CV
2 CV
2 CV
4 CV
0.25
4/6
0.5
0.5
0.20
3.2/4.8
0.4
0.4
Deionized water A5
GST A-buffer
A1
10 CV
5 CV
1/5
1/5
0.8/4
0.8/4
Deionized water
Deionized water
Deionized water
Deionized water
A5
A5
A5
A5
4 CV
2 CV
2 CV
2 CV
0.25
4/6
0.5
0.5
0.20
3.2/4.8
0.4
0.4
A5
A5
A5
A5
10 CV
4 CV
2 CV
2 CV
1/5
0.25
4/6
0.5
0.8/4
0.20
3.2/4.8
0.4
2 CV
2 CV
4/6
0.5
3.2/4.8
0.4
4 CV
5 CV
4 CV
4 CV
Step 3
Strip
HisTrap HP, HiTrap Chelating
(1 ml / 5 ml)
GSTrap HP/FF (1 ml / 5 ml)
HiTrap Desalting
HiPrep 26/10 Desalting
HiTrap Q/SP HP
RESOURCE Q/S (1 ml / 6 ml)
Mono Q/S
HiLoad Superdex 75/200 pg
0.8/4
30% 2-Propanol
1% Triton X-1004)
Deionized water
Deionized water
1M NaOH
1M NaOH
1M NaOH
30% 2-Propanol
Step 4
A3
A7
A5
A5
A7
A7
A7
A3
10 CV
4 CV
5 CV
5 CV
4 CV
5 CV
4 CV
0.5 CV
0.5/2.5
1/5
7.5
15
0.25
4/6
0.5
0.5
0.4/2
0.8/4
6
12
0.20
3.2/4.8
0.4
0.4
Step 5
HisTrap HP, HiTrap Chelating
(1 ml / 5 ml)
HiTrap Q/SP HP
RESOURCE Q/S (1 ml / 6 ml)
Mono Q/S
2M NaCl
30% 2-Propanol
1M HCl
1M HCl
Step 7
RESOURCE Q/S (1 ml / 6 ml) 1M NaCl
Mono Q/S
1M NaCl
Step 6
B2
A3
A1
A1
1 CV
2 CV
5 CV
4 CV
1/5
0.25
4/6
0.5
0.8/4
0.20
3.2/4.8
0.4
Deionized water
Deionized water
Deionized water
Deionized water
B1
B1
5 CV
4 CV
4/6
0.5
3.2/4.8
0.4
Step 8
Deionized water A5
Deionized water A5
Step 1 implies that the column is already filled with deionized water.
If this is not the case, an additional step using water has to be added.
1)
2)
3)
4)
cue 18-1178-03 AA 2004-03 • p26
DS
CIP Regular, recommended solutions
cue 18-1178-03 AA 2004-03 • p27
Pause 1 hour.
Pause over night at RT.
Alternatively 3–4 CV 70% ethanol.
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Automated multi-step protein purification
Automated multi-step protein purification
Advanced zone
Advanced zone
General
In the Advanced zone,
all main parameter
Advanced zone
values can be viewed,
and modified if required.
Examples of parameters that may
be modified:
• Wash volumes
• Flow rates
• Gradient slopes
• Watch parameters for peak detection
Note: Do not change default parameter values in a
method plan unless the result is clearly
understood. Changing one parameter value
understood
might require adjustments of other parameter
values as well.
Tip
Use the Note field in the Save As
page to specify changes made to
the default settings.
Advanced zone, continued
Some considerations
in Advanced Zone
Flow rates and pressure limits
No warning will be issued if the flow rate or pressure limit
are set higher than the values recommended for the
columns used.
Note: Changing the flow rate will change the slope of an
eluting peak.
Peak detection and collection
The default values for detecting peak start and peak end
are set to match typical peaks eluting from the supported
columns. Different values are used for normal and high
sample loading. The slope and levels values though
might need to be adjusted if other parameters are
changed.
Example of peak collection in loops:
UV max
UV max'
The slope of a peak is changed if:
2
• The flow rate is changed, or
2'
• The gradient slope is changed.
2:nd wash of AC chelating column
A second wash of affinity chelating columns can be used
to wash out loosely bound proteins. The second wash is
an additional step before the elution of the target protein.
For a powerful wash, the imizadole concentration %B
should be set to above 0%B. If no imidazole is used
(%B=0), the volume used in this step should be
decreased to minimize the run time.
Note: Do not raise the imidazole concentration too
much as it might cause the target protein to come
off as well.
Note: The second wash is by default not included for
GSTrap columns.
Peak volume in AC and IEX elution steps
Peak volumes for peaks eluted from affinity and ion
exchange columns should be as small as possible. It will
increase the possibility to:
• Load the entire peak volume onto any desalting or gel
filtration column in the next step since their maximum
loading volumes are limited.
• Optimize the recovery in the ion exchange step by
making sure that the entire peak volume is collected
when eluted in the ion exchange gradient. Only one
loop is available for each peak, so peak volumes larger
than 7.5 ml will cause sample loss.
To minimize the peak volume:
• Choose a suitable column type.
• Choose a suitable column size.
• Adjust the gradient slope. A steeper gradient will
narrow the peaks.
Protease injection volume
This applies to protocols that include tag cleavage.
If necessary, change Watch Slope Greater than.
1'
3'
1
3
The default level values might need adjustment if:
• Small peaks are expected, or
• The peaks are broadened due to e.g. a more flat
gradient.
If necessary, change Watch Level Greater than and Watch
Level Less than.
1
Greater_Than AND Slope_Greater_Than
2
Peak_Max with Factor 0.6
3
Less_Than_or_Valley OR Stable_Plateau
Note: If the level values are decreased for very large or
broad peaks, the peak volume collected might be
too big for the loop available or exceed the
loading volume of the next column.
Watch instructions for peak collection
Greater_Than The signal exceeds a certain value.
Peak fractionation parameters
The parameters are based on slope or level.
Example of peak fractionation in microplate:
Less_Than The signal falls below a certain value.
UV max
Slope_Greater_Than The slope of the the signal curve exceeds
a specified value.
Slope_Less_Than The slope of the the signal curve falls below
a specified value. It is always used in combination with
Peak_Max.
3
Less_Than_Or_Valley The signal falls below a specified value
or a valley is detected. A valley is detected only after a
Peak_Max has been detected, and the valley is defined by a
local minimum followed by an increase to 102% of the local
minimum plus the Delta_Peak value.
Peak_Max The signal falls to a specified fraction of the most
recent peak maximum minus the Delta_Peak value.
Stable_Baseline The signal is stable within the limits of the
Delta_Base value for a specified time interval.
Stable_Plateau The signal is stable within the limits of the
Delta_Plat value for a specified time interval.
The default value for the protease injection is 0.7 CV. If
using a larger value, the target protein might be lost
since it will pass through the column before peak
collection is activated.
cue 18-1178-03 AA 2004-03 • p28
Intermediate peak collection parameters
cue 18-1178-03 AA 2004-03 • p29
1
2
4
1
5
2
4
Start Slope OR Start Level
UV max × Peak Max Factor 0.5
3
5
Stop Level OR Stop Slope
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Automated multi-step protein purification
Automated multi-step protein purification
Flow charts
Flow charts, continued
• Valve positions after reset (END), no flow
• Column equilibration
• Sample loading
Waste
Frac
• Wash out unbound sample
• Elution
or
Waste
F3
A
H
Outlet Valve
F3
F4
F11
Frac
F4
F11
F10
A
H
Outlet Valve
F5
F5
F6
F9
F10
F7
F8
F6
F9
Fraction Collector
F7
F8
Fraction Collector
OutV
OutV
0.2 MPa
Loop
L1
Restr
L3
L2
L4
0.2 MPa
Loop
L5
C3in
C5in
C3
C4
C2
Bypass
L4
BP
C5in
L2
UV
Bypass
L4
L5
C5
C3
C4
C1
C2
C4
C3
L5
L2
L4
L3
L2
L1
Loop Valve
C5out
C2out
C4out
C3out
Manual
inject
Waste
C1
Waste
Column Valve
L4
L5
L3
UV
C1out
ColV
BP
L1
BP
Bypass
BP
LoopV2
C5
Bypass
L1
C4out
Manual
inject
C1
L5
L2
Loop Valve
C3out
C2
Cond
UV
L3
UV
C2out
L4
L1
BP
C1in
L3
C5out
C1out
Cond
C2in
L1
L5
BP
BP
C4in
Cond
C1
L3
L2
C3in
L1
BP
C1in
C5
L1
Restr
Cond
C2in
C4in
C2
C4
C3
LoopV2
C5
Waste
ColV
Waste
Column Valve
Injection Valve
Injection Valve
MixInj
MixInj
P
A8
Inlet Valve
P
S4
A7
Press
A6
A5
Mix2
Mixer
S2
A4
Mix1
A8
S3
S1
IN
IN
NC
NO
A2
NC
A1
Samples
NO
B1
IN
B2
SW2
IN
SW1
SW2
SW1
NC
Samples
NO
B1
A2
SW2
AirS
Pump
Buffers
Pump
Air sensor
AirS
Pump
Buffers
Pump
cue 18-1178-03 AA 2004-03 • p30
S1
A3
SW1
SW2
SW1
S2
A4
Mix1
B2
S3
A5
Mix2
Mixer
NC
A1
S4
A6
A3
NO
Inlet Valve
A7
Press
cue 18-1178-03 AA 2004-03 • p31
Air sensor
cue card
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Automated multi-step protein purification
Automated multi-step protein purification
Flow charts, continued
Flow charts, continued
• Intermediate fractionation
• System wash
• Pump wash
Waste
Waste
Frac
F3
F3
A
H
Outlet Valve
Frac
F4
F4
F11
F11
F5
F5
F10
F10
F6
F6
F9
F9
F7
F7
F8
F8
Fraction Collector
Fraction Collector
OutV
OutV
Loop
0.2 MPa
0.2 MPa
Loop
L1
Restr
L3
L2
L4
L5
Cond
C2in
C4in
C3
C4
C2
L1
Cond
C1
L4
BP
Bypass
C5in
C5
C3
C4
C2
L4
L5
L3
L2
L1
C1
Waste
ColV
L2
L1
Loop Valve
C4out
C3out
C2
Manual
inject
C4
C3
LoopV2
C5
Waste
ColV
Waste
Column Valve
L3
C5out
C1out
LoopV2
C5
L4
L5
L3
UV
C2out
Manual
inject
C4
C3
L2
L4
BP
Bypass
BP
BP
L1
L5
Loop Valve
C3out
Bypass
UV
C4out
C2
L5
L1
L3
C5out
C1out
C1
L4
Cond
C1
UV
C2out
L3
L2
BP
C1in
L2
UV
BP
BP
L1
L5
Cond
C2in
C4in
Bypass
BP
C1in
C5
L1
Restr
C3in
C3in
C5in
A
H
Outlet Valve
Waste
Column Valve
Injection Valve
Injection Valve
MixInj
MixInj
P
P
A8
A8
Inlet Valve
A6
A5
Mix2
Mixer
S3
S2
A4
Mix1
S1
IN
Mix1
NC
A2
A1
Samples
IN
Buffers
IN
SW2
Air sensor
Samples
NO
A2
SW2
AirS
Pump
Buffers
Pump
cue 18-1178-03 AA 2004-03 • p33
SW1
SW2
NC
Pump
cue 18-1178-03 AA 2004-03 • p32
B2
SW1
B1
AirS
Pump
S1
A3
NO
B1
S2
NC
SW1
SW2
S3
A5
Mix2
Mixer
NO
B2
SW1
S4
A6
NC
A1
IN
Press
A4
A3
NO
Inlet Valve
A7
S4
A7
Press
Air sensor
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Automated multi-step protein purification
Automated multi-step protein purification
Flow charts, continued
• Sample loading from loops
• Loop wash
Waste
Frac
F3
A
H
Outlet Valve
F4
F11
F5
F10
F6
F9
F7
F8
Fraction Collector
OutV
0.2 MPa
Loop
L1
Restr
L3
L2
L4
L5
C3in
C5in
Cond
C2in
C4in
C5
C3
C4
C2
L1
Bypass
BP
C1in
Cond
C1
L5
L2
UV
L4
BP
Bypass
BP
BP
L1
L4
L5
L3
L3
L2
L1
UV
Loop Valve
C5out
C1out
C2out
C1
C4out
C3out
C2
Manual
inject
C4
C3
LoopV2
C5
Waste
ColV
Waste
Column Valve
7
Injection Valve
MixInj
P
A8
Inlet Valve
S4
A7
Press
A6
A5
Mix2
Mixer
S3
S2
A4
Mix1
S1
A3
NO
NC
A1
IN
B2
IN
SW1
SW2
SW1
NC
Samples
NO
B1
A2
SW2
AirS
Pump
Buffers
Air sensor
Pump
cue 18-1178-03 AA 2004-03 • p34
cue 18-1178-03 AA 2004-03 • p35
cue card
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Automated multi-step protein purification
Automated multi-step protein purification
Reference information
Overview of columns and solutions
Protocol
A
• Flow overview, simplified
B
C
D
E
F
G
Column position
1
AC(1)
AC(1)
AC(1)
AC(1)
AC(1)
–
–
2
AC(2)
AC(2)
AC(2)
AC(2)
AC(2)
–
–
3
AC(3)
AC(3)
AC(3)
AC(3)
DS
–
–
4
AC(4)
AC(4)
IEX
IEX
IEX
–
–
5
GF
DS
DS
DS
GF
DS
GF
outlet
valve
A1
AC binding
AC binding
AC binding
AC binding
AC binding
–
–
B1
AC wash
AC wash
AC wash
AC wash
AC wash
–
–
A2
–
–
IEX binding1)
IEX binding
IEX binding1)
–
–
B2
–
–
IEX elution
IEX elution
IEX elution
–
–
A3
AC elution
AC elution
AC elution
AC elution
AC elution
–
–
A4
GF buffer
DS buffer
–
DS buffer
GF buffer
A5
–
–
–
–
–
A6
A7
0.5 M NaOH2) 0.5 M NaOH2) 0.5 M NaOH2)
0.5 M NaOH2) 0.5 M NaOH2)
flow
through,
sample 1–4
not used
peaks,
sample 1–4
Cond.
Cell
loops
1-5
loop
valve
UV cell
DS buffer GF buffer
–
–
–
–
Cleavage
Cleavage
Cleavage
Cleavage
Cleavage
–
buffer3)
buffer3)
buffer3)
buffer3)
buffer3)
–
–
–
–
–
–
–
–
–
S1
Sample 1
Sample 1
Sample 1
Sample 1
Sample 1
S2
Sample 2
Sample 2
Sample 2
Sample 2
Sample 2
Sample 2 Sample 2
S3
Sample 3
Sample 3
Sample 3
Sample 3
–
Sample 3 Sample 3
S4
Sample 4
Sample 4
–
–
–
Sample 4 Sample 4
A8
0.2
MPa
waste,
metal ion
waste
Buffer inlet
96-well microplate
column
valve
alternative :
superloop
columns
1-5
Sample inlet
1)
2)
3)
Sample 1 Sample 1
mixer
injection
valve
pressure
sensor
The IEX binding buffer is also used for desalting.
If NaOH wash between samples is chosen.
If Tag cleavage and Fill columns with cleavage buffer
are chosen.
waste
inlet
valve
pump
gradient
valves
air sensor
cue 18-1178-03 AA 2004-03 • p36
cue 18-1178-03 AA 2004-03 • p37
buffers
A1, B1
buffers
A2, B2
buffers
A3-8
Samples
1-4
cue card
Automated multi-step protein purification
cue 18-1178-03 AA 2004-03 • p38
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Automated multi-step protein purification
cue 18-1178-03 AA 2004-03 • p39
cue card
Automated multi-step protein purification
Ordering information
Product
Pack Size
Code Number
Product
ÄKTAxpress module, system extension 1
18-6645-01
Ion exchange chromatography
RESOURCE Q
1 x 1 ml
ÄKTAxpress, set of 4 systems
18-6645-04
RESOURCE Q
1 x 6 ml
17-1179-01
RESOURCE S
1 x 1 ml
17-1178-01
17-1180-01
Systems
1
Pack Size
Code Number
17-1177-01
ÄKTAxpress, set of 4 systems
with computer
1
18-6645-05
RESOURCE S
1 x 6 ml
Flask holder
1
18-1177-79
HiTrap Q HP
5 x 1 ml
17-1153-01
HiTrap SP HP
5 x 1 ml
17-1151-01
Supported columns
Mono Q 5/50 GL
1 x 1 ml
17-5166-01
HisTrap HP
5 x 1 ml
17-5247-01
Mono S 5/50 GL
1 x 1 ml
17-5168-01
HisTrap HP
100 x 1 ml
17-5247-05
Gel filtration
HiLoad 16/60 Superdex 75
prep grade
1 x 120 ml
17-1068-01
HiLoad 16/60 Superdex 200
prep grade
1 x 120 ml
17-1069-01
Affinity chromatography
HisTrap HP
5 x 5 ml
17-5248-02
HisTrap HP
100 x 5 ml
17-5248-05
HiTrap Chelating HP
5 x 1 ml
17-0408-01
HiTrap Chelating HP
5 x 5 ml
17-0409-03
HiTrap Chelating HP
100 x 5 ml
17-0409-05
Superloop
GSTrap HP
5 x 1 ml
17-5281-01
Superloop
GSTrap HP
100 x 1 ml
17-5281-05
Documents
GSTrap HP
5 x 5 ml
17-5282-02
UNICORN manual set
1
11-0003-68
GSTrap HP
100 x 5 ml
17-5282-05
ÄKTAxpress User Manual
1
18-1178-00
18-1178-02
1
18-1113-82
GSTrap FF
5 x 1 ml
17-5130-01
ÄKTAxpress Safety Handbook
1
GSTrap FF
100 x 1 ml
17-5130-05
ÄKTAxpress Installation Guide
1
18-1178-01
GSTrap FF
5 x 5 ml
17-5131-02
HiTrap Column Guide
1
18-1129-81
GSTrap FF
100 x 5 ml
17-5131-05
Affinity Chromatography Handbook
1
18-1022-29
Gel Filtration Handbook
1
18-1022-18
Desalting
HiPrep 26/10 Desalting
1 x 53 ml
17-5087-01
Ion Exchange Chromatogr. Handbook 1
18-1114-21
HiPrep 26/10 Desalting
18-1132-29
4 x 53 ml
17-5087-02
Protein Purification Handbook
1
HiTrap Desalting
5 x 5 ml
17-1408-01
Recombinant Protein Handbook
1
18-1142-75
HiTrap Desalting
100 x 5 ml
11-0003-29
GST Gene Fusion System Handbook
1
18-1157-58
100-packs are special packs delivered on customer order.
Includes connector package, domed nuts and instructions.
Code numbers for accessories and user replaceable spare parts
can be found in the ÄKTAxpress User Manual.
Asia Pacific Tel: +852 2811 8693 Fax: +852 2811 5251 Australasia Tel: +61 2 9899 0999 Fax: +61 2 9899 7511 Austria Tel: 01 57 606 16 19
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Tel: +43 1 982 3826 Fax: +43 1 985 8327 Denmark Tel: 45 16 2400 Fax: 45 16 2424 Finland & Baltics Tel: +358 (0)9 512 3940
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10 Switzerland Tel: 0848 8028 12 Fax: 0848 8028 13 UK Tel: 0800 616 928 Fax: 0800 616 927 USA Tel: +1 800 526 3593 Fax: +1 877 295 8102
UNICORN, ÄKTAxpress, HiTrap, HisTrap, GSTrap, HiLoad, HiPrep, RESOURCE, Superdex, Mono Q, Mono S, Superloop, PreScission and Drop Design are
are trademarks of Amersham Biosciences Limited. Amersham and Amersham Biosciences are trademarks of Amersham plc. Amersham Biosciences AB
Björkgatan 30, SE-751 84 Uppsala, Sweden. Amersham Biosciences UK Limited Little Chalfont, Buckinghamshire, England HP7 9NA. Amersham Biosciences Corporation 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855 USA. Amersham Biosciences Europe GmbH Munzinger Strasse 9, D79111 Freiburg, Germany. Amersham Biosciences KK Sanken Building, 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan. All goods and
services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. A copy of these
terms and conditions is available on request.
© Amersham Biosciences AB 2004 – All rights reserved.
cue 18-1178-03 AA 2004-03 • p40