Bio-Rad Nuvia™ IMAC Resin - Manual (Page 27)

Nuvia IMAC Ni-Charged Resin 23
8. Equilibrate the column with 10 column volumes of binding
buffer (0%B). Stop collecting fractions.
9. Identify the fractions that contain the histidine-tagged protein
using an activity assay (such as one of Bio-Rad’s protein assay
kits), UV absorbance, SDS-PAGE, or western blot analysis
with antihistidine antibodies or antibodies specific to the target
protein.
10. Calculate the concentration of imidazole that corresponds to
the elution peak of the histidine-tagged protein.
Note: The delay due to column volume and the tubing in
the chromatography system need to be considered when
comparing the actual gradient trace to the programmed
gradient.
11. Based on calculated imidazole concentration, a stepwise
experiment may now be designed.
The following tips are useful to keep in mind when designing the
experiment:
• Maintain the concentration of imidazole in the binding
(also called equilibration) buffer at 20 mM. If large amounts
of contaminants are also adsorbed onto the resin, the
concentration of imidazole in the sample and equilibration
buffer may be increased. This may reduce the overall amount
of target protein bound and should be carried out with care.
However, it will also increase the column’s binding capacity
for the target protein due to the reduction in contaminating
proteins
• Include a wash step with an imidazole concentration slightly
lower than the concentration necessary to elute the target
protein. This will increase purity by removing unbound
contaminants without eluting the bound histidine-tagged
protein. The optimized wash step should include 50 mM
sodium phosphate, 0.3 M NaCl, and an appropriate
concentration of imidazole
• The elution buffer should contain a concentration of imidazole
greater than the calculated concentration corresponding to the
eluted peak of target protein
• Perform a trial run