Bio-Rad Nuvia™ IMAC Resin - Manual (Page 22)

18 Nuvia IMAC Ni-Charged Resin
2. Equipment
• IMAC column (as prepared in Section 4)
3. Biological Sample
• Clarified lysate
The binding capacity of the Nuvia
™ IMAC resin is ~40 mg
histidine-tagged protein per ml resin. Larger amounts of
protein will require use of a larger column.
4. Additional Materials
• Medium-pressure chromatography system (such as
Bio-Rad’s BioLogic ™ or NGC ™ system)
• Equipment for determining total protein concentration
within the lysate
Method
1. Equilibrate the column with at least 5 column volumes of
binding buffer.
2. Add or dilute sample in binding buffer and load onto the
column using a desired flow rate.
The choice of binding buffer will vary based on the properties
of the sample to be purified. Sodium or potassium phosphate
is recommended as a general starting buffer; for example,
50 mM sodium phosphate, 300 mM NaCl, 5 mM imidazole,
pH 8.0. Binding of histidine-tagged protein on the Nuvia IMAC
resin is optimal in the pH range of 7–8.
The column may be run at flow rates up to 500 cm/hr. Higher
binding of histidine-tagged proteins will be achieved at lower
flow rates. Average binding capacity of the Nuvia IMAC resin is
approximately 40 mg histidine-tagged protein/ml resin.
3. Collect fractions.
These fractions represent unbound proteins.
4. Wash the resin with at least 5 column volumes of wash buffer
to remove unbound sample.
Wash out remaining unbound solutes. Repeat wash steps as
necessary for the A
280
to be at or near baseline.