Bio-Rad Profinity IMAC Resins User Manual
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The following are useful items to keep in mind during the design of the
experiment:
•
Maintain the concentration of imidazole in the binding (also called
equilibration) buffer at 20 mM. If high amounts of contaminants are also
adsorbed onto the resin, the concentration of imidazole in the sample
and equilibration buffer may be increased. This measure might reduce the
overall amount of target protein bound and should be carried out with
care. However, it will also increase the column binding capacity for the
target protein due to a reduction of contaminating proteins.
•
Include a wash step containing 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 His-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 (see Section 11).
Part 2: Using an Optimized Imidazole Concentration for Purification
1.
Prepare 200 ml binding buffer and 200 ml elution buffer for a 1 ml
column.
Purge the pumps with the fresh buffers. Use suitable buffer volumes for larger
scale purifications.
2.
Equilibrate the column with 10 column volumes of equilibration/binding
buffer.
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 column with a minimum of 5 column volumes of binding buffer
to remove unbound contaminants.
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