GE AKTApure User Manual page 443

Predefined purification
method
NHS-coupling
ÄKTA pure User Manual 29119969 AB
Principle
A column packed with NHS-activated Sepharose
is washed with 1 mM HCl, followed by immediate
application of the protein for covalent coupling
onto the column. After incubation the non-bound
protein is washed out and the remaining active
groups are deactivated with ethanolamine
buffer, followed by further washes.
Required components
The required component for this method is an
Inlet valve (2-ports or 7-ports).
Required solutions
The method phases are preconfigured to use
the following solutions in the following inlets and
sample loop:
Inlet A1: Coupling buffer, for example 0.2 M
NaHCO + 0.5 M NaCl pH 8.3.
3
Inlet A2: Activation solution, for example 1 mM
HCl
Inlet B1: High pH buffer, for example 0.5 M
ethanolamine, 0.5 M NaCl pH 8.3
Inlet B2: Low pH buffer, for example 0.1 M Sodi-
um acetate, 0.5 M NaCl pH 4.0
Sample loop: Ligand in coupling buffer
Note:
The NHS coupling phase is preconfigured to
empty the loop with the ligand with a volume of
1 ml. The recommended ligand volume is 1 CV
(column volume). If a column volume different
than 1 ml is used, this value needs to be changed
to correspond to 1 CV.
9 Reference information
9.7 Predefined methods and phases
9.7.1 Predefined purification methods
Included phases
443