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On-column refolding
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):
6 M guanidine hydrochloride, 20 mM Tris-HCl, 0.5 M NaCl,
5 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0
Solubilisation buffer (port A2):
6 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole,
1 mM 2-mercaptoethanol, pH 8.0
Elution buffer (port A3):
20 mM Tris-HCl, 0.5 M NaCl, 0.5 M imidazole,
1 mM 2-mercaptoethanol, pH 8.0
Refolding buffer (port B):
20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole,
1 mM 2-mercaptoethanol, pH 8.0
Prepare at least 500 ml of each eluent.
* Alternative binding buffers:
5–40 mM imidazole can be included in the binding buffer to
reduce unspecific 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.
** Include the same imidazole concentration as in used binding
buffer.
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 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 40) 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.
11-0027-48 AC, 2007-09 • p14
*
**
**
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
prime
should be displayed.
b) Use the arrow and OK buttons to move in the menu
tree until you find
On-Column Refolding
Templates
Application Template
On-Column Refolding
HisTrap
c)
Enter the sample volume and press
template.
Note: If a 5 ml column is preferred see cue card on
p.36.
On-column Refolding HisTrap
Theoretical gradient in
Template.
%B
100
Refolding
Priming
Buffer wash
50
& Priming
Equili-
bration
Sample
2 10
30
Total separation time = 160 min + sample application time
Controlled By:
HisTrap.
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
60
20
20
17
Min
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