Bio-Rad Nuvia™ IMAC Resin - Manual (Page 26)

22 Nuvia IMAC Ni-Charged Resin
3. Biological Sample
• Clarified lysate
Note: Keep the sample as small as possible during
optimization of binding and elution conditions.
4. Additional Materials
• Equipment for assessing protein purity and recovering of
the histidine-tagged protein
Method
Part 1: Optimizing the Imidazole Concentration
1. Purge the entire flow path of the chromatography system with
water according to the manufacturer’s instructions.
Connect the column and wash it with 10 column volumes
of water. Disconnect the column either by valve switching
or manually. Purge the flow path before the column with
elution buffer from inlet B and, with the column offline, then
with binding buffer from inlet A. Purge the entire system with
binding buffer. Reconnect the column to the system.
2. Equilibrate the column with 10 column volumes of binding
buffer (0%B).
3. Begin collecting fractions of 1 column volume.
If a 1 ml IMAC column is used, 1 ml fractions are
recommended. For larger columns, reduce the fractions
collected to amounts 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 sample is being applied.
If the sample is insufficiently clarified, backpressure will
increase.
5. Wash the unbound material with 10 column volumes of
binding buffer (0%B).
6. Elute the sample with a linear gradient of 0–50% elution buffer.
7. Wash the column with 100% of the elution buffer for 5 column
volumes.