GE HEALTHCARE AKTAprime plus, Quick Reference Instructions

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11-0027-48 AC, 2007-09 • p15
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
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
* 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 filtered through a 0.45 µm filter.
•
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 buffer is
connected to the correct inlet port.
•
Check that the composition and pH of the buffers are
correct.
•
Check that the sample has been adjusted to binding
buffer conditions.
•
Check that your sample contains target protein.
No elution:
•
Check that the inlet tubing from each buffer is
connected to the correct inlet port.
•
Check that the composition and pH of the buffers are
correct.
•
Use alternative elution conditions according to the
column instructions.
•
Check that your sample contains target protein.
Sample: Clarified homogenate of E. coli expressing
histidine-tagged protein
Column: HisTrap FF 1 ml
Binding buffer (A1): 6 M Guanidine hydrochloride, 20 mM
Tris-HCl, 0.5 M NaCl, 5 mM Imidazole, 1mM
2-mercaptoethanol, pH 8.0
Solubilization 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
20 40 60 80 100 120 140 min
0.4
0.3
0.2
0.1
0
UV 280 nm
Programmed %B
AU
280 nm
100
80
60
40
20
0
% B
5 Typical result