Bio-Rad Nuvia™ IMAC Resin - Manual (Page 28)

24 Nuvia IMAC Ni-Charged Resin
Part 2: Using an Optimized Imidazole Concentration for
Purification
1. Prepare 500 ml binding buffer and 500 ml elution buffer for a 5
ml column.
Purge the pumps with the fresh buffers. Adjust buffer volumes
for larger scale purifications.
2. Equilibrate the column with 10 column volumes of
equilibration/binding buffer.
3. Begin collecting fractions.
If a 1 ml IMAC column is used, 1 ml fractions are
recommended. For larger columns, reduce the fractions
collected to amounts ranging from 0.2 to 0.5 column volumes.
4. Load sample and collect the flowthrough in fractions
appropriate to the size of the column, as recommended
above.
Note: Monitor the backpressure while the sample is
being applied. If the sample is insufficiently clarified, the
backpressure will increase.
5. Wash the column with a minimum of 5 column volumes of
binding buffer to remove unbound contaminants.
6. Wash the column with a minimum of 5 column volumes of
binding or starting buffer that contains imidazole in a quantity
that will not elute the target protein.
7. Elute the histidine-tagged protein with 5 column volumes of
elution buffer with an optimized imidazole concentration.
8. Wash the column with 5 column volumes of elution buffer.
Stop collecting fractions.
9. Re-equilibrate the column with 10 column volumes of
equilibration (binding) buffer.
10. Assess the purity and recovery of the target 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.