Bio-Rad Profinity IMAC Resins - Manual (Page 39)

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Biological Sample
• Denatured protein sample as prepared in Section 8
Additional Materials
• Equipment for assessing protein purity and recovery of the His-tagged
protein
Method
Reserve a small amount of lysate (as prepared in Section 8) 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 2 ml spin collection tube.
3. Preequilibrate the spin column with 5 column volumes of binding buffer.
4. Add an appropriate amount of lysate ( ≤0.5 ml) to the micro spin column.
See Section 8, Preparation of Clarified E. coli Lysate Using Denaturing
Conditions for protocol.
5. Mix by pipetting up and down 5 times.
Incubate in micro spin column for up to 5 min.
6. Centrifuge at 1,000 x g (700 x g for 2 min) for 15 sec.
Remove the unbound proteins by centrifuging. Save flow-through and label it
“unbound 1”.
Part 2: Washing the Resin
7. Insert micro spin column into new collection vessel.
8. Wash the resin with at least 5 column volumes of wash buffer.
Pipet up and down at least 5 times.
Note: If previously determined, an optimized concentration of imidazole may
be added to the contents of the guanidine binding buffer that is slightly less
than the concentration necessary to elute the target protein. See Section 10.
9. Centrifuge at 1,000 x g for 15 sec (or 700 x g for 2 min).
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