Bio-Rad Nuvia™ IMAC Resin - Manual (Page 32)

28 Nuvia IMAC Ni-Charged Resin
Section 10
Regenerating, Cleaning, Sanitizing, and Storage
Nuvia ™ 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 they need to be recharged with metal ions. When it
becomes necessary, regenerate metal-charged Nuvia IMAC resins
by first 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 if 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 can be run at 2–5 ml/min.