Bio-Rad Nuvia™ IMAC Resin - Manual (Page 30)

26 Nuvia IMAC Ni-Charged Resin
3. Biological Sample
• Clarified lysate
The binding capacity of Nuvia
™ IMAC resin is ≥40 mg histidine-
tagged protein per ml resin. Larger amounts of protein will
require a larger column
4. Additional Materials
• Equipment for assessing protein purity and recovery of the
histidine-tagged protein
Method
Reserve a small amount of lysate prior to loading sample onto
the column. This will serve as sample for the lysate lane for later
analysis with, for example, SDS-PAGE.
Part 1: Binding the Sample
1. Start with a prepacked spin column, charged with the metal
ion of choice.
See Section 5, Column Packing — Sample Preparation–Sized
Columns for protocol.
2. Place prepacked spin column in an appropriate spin collection
tube.
3. Pre-equilibrate the spin column with 5 column volumes of
binding buffer.
The choice of binding buffer will vary based on the properties
of the sample to be purified. Potassium phosphate and
sodium phosphate are recommended as general starting
buffers; for example, 50 mM sodium or potassium phosphate,
300 mM NaCl, pH 8.0. Binding of histidine-tagged protein on
the Nuvia
™ IMAC resin is optimal in the pH range of 7–8.
4. Add an appropriate amount of lysate (≤0.5 ml) to the micro
spin column.
5. Mix by pipetting up and down 5 times.
Incubate for up to 5 min in micro spin column.
6. Centrifuge at 1,000 x g for 1 min.
Remove the unbound proteins by centrifuging.