Bio-Rad Profinity IMAC Resins - Manual (Page 44)

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Section 15
Regenerating, Cleaning, Sanitizing, and
Storage
Profinity ™ IMAC columns are well suited for reuse. The polymeric nature and open
pore structure of the resin allow the column to be run at high flow rates during
regeneration, cleaning, and sanitizing steps. Protein separations are unaffected,
even after numerous cycles, as reproducibility is extremely high.
Unless otherwise stated, the following steps may be carried out at 2 ml/min.
Regenerating the Medium
Regeneration cleans the medium adequately to start the next cycle. In general,
IMAC columns may be used a number of times before it becomes necessary to
recharge them with metal ions. If this is the case, regenerate metal-charged
Profinity IMAC resins by stripping with an EDTA solution. Wash the column with
10 column volumes of 20 mM sodium phosphate, up to 1 M NaCl, and 0.2 M
EDTA, pH 7.4. Ensure that residual EDTA is completely removed from the column
by washing it with 3–5 column volumes of binding buffer followed by 3–5 column
volumes of distilled water.
Recharge the IMAC resin as recommended in Section 6, Immobilizing Metal Ions,
or proceed with cleaning-in-place measures (below).
Cleaning-in-Place
A column used to purify protein from cell extract usually contains some soluble
substances and cell debris that are nonspecifically adsorbed onto the matrix.
Cleaning-in-place eliminates material not removed by regeneration and prevents
progressive buildup of contaminants. If the column is to be reused, these
contaminants should be cleaned from the column, as they were not completely
removed during the sample clarification steps.
The following steps may be followed to clean IMAC columns. This protocol also
includes a regeneration step. For optimal results, the column should be run at
2 ml/min.
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