Bio-Rad Profinity IMAC Resins - Manual (Page 28)

24
Method
Part 1: Optimizing the Imidazole Concentration
1. Purge the entire flow path of the chromatography system with water
according to manufacturer’s instructions.
Connect the column and wash it with 10 column volumes of water.
Disconnect the column by valve switching or manually. Purge the flow path
before the column with elution buffer from inlet B, and with the column off the
line, then with binding buffer from inlet A. Purge the entire system with binding
buffer. Reconnect the column to the system.
2. Equilibrate 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 ranging from
0.2 to 0.5 column volumes.
4. Load sample and collect the flow-through 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 unbound material with 10 column volumes of binding buffer
(0% B).
6. Elute the sample with a linear gradient of 0% to 50% elution buffer.
7. Wash the column with 100% of the elution buffer for 5 column volumes.
8. Equilibrate the column with 10 column volumes of binding buffer
(0% B). Stop collecting fractions.
9. Identify the fractions containing the His-tagged protein.
Use an activity assay (Bio-Rad’s protein assay kit), UV absorbance,
SDS-PAGE, or western blot analysis with anti-histidine antibodies or
antibodies specific to the target protein.
10. Calculate the concentration of imidazole corresponding to the elution
peak of the His-tagged protein.
Note: Delay due to the column dead volume and chromatography system
needs to be considered compared to the programmed gradient.
11. Based on calculated imidazole concentration, a stepwise experiment
may now be designed.
10001677B.qxd 1/28/2005 12:42 PM Page 27