Bio-Rad Profinity IMAC Resins - Manual (Page 36)

32
Method
Reserve a small amount of lysate (as prepared in Section 7) prior to loading sample
onto the column. This will serve as sample for the “lysate” lane for later analysis, for
example, SDS-PAGE.
Part 1: Binding of 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. Preequilibrate 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 His-tagged protein on the
Profinity
™ 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.
Part 2: Washing the Resin
7. Insert micro spin into new collection vessel.
8. Wash the resin with at least 5 column volumes of binding buffer
containing imidazole.
Pipet up and down at least 5 times.
Note: If previously determined, an optimized concentration of imidazole may
be used that is slightly less than the concentration necessary to elute the
target protein. See Section 10.
9. Centrifuge at 1,000 x g for 1 min.
Remove remaining unbound proteins by centrifuging. The wash step can be
repeated if necessary.
10001677B.qxd 1/28/2005 12:42 PM Page 35