Bio-Rad Profinity IMAC Resins - Manual (Page 42)

38
Additional Materials
• Equipment for assessing protein purity and recovery of the His-tagged
protein
Resin Preparation
Profinity IMAC and Profinity IMAC Ni-charged resins come supplied in a
20% ethanol solution for resin storage. Before purification, the resin storage
solution provided must first be replaced with distilled water.
1. For easy removal of storage solution, transfer an appropriate amount of
resin slurry to a graduated container, such as a Bio-Rad Econo-Pac
column (bed volume up to 20 ml).
2. Apply vacuum to the column for rapid removal of the storage solution.
Exert caution to not allow the resin to dry out.
3. Wash the column with 3 column volumes of distilled water.
4. Add enough distilled water to make a 50% slurry.
5. The resin is now ready to be packed.
Method
1. Prepare the resin.
If using uncharged Profinity IMAC resin, charge the resin as recommended in
Section 6, Immobilizing Metal Ions.
If using Profinity IMAC Ni-charged resin, ensure that all ethanol has been
thoroughly washed away before proceeding to step 2.
2. Add an appropriate amount of the prepared resin slurry (see Resin
Preparation above) to an appropriate amount of clarified lysate.
3. Swirl mixture gently in an appropriate container. Incubate the resin-lysate
mixture at 4°C for up to 30 min.
4. Load the resin-lysate mixture into an appropriate-sized column. Cap the
bottom outlet of the column.
5. Collect column flow-through.
Save the flow-through for SDS-PAGE analysis.
6. Wash column with at least 5 column volumes of binding/washing buffer.
Collect wash fractions. Pool with fractions collected in step 5.
10001677B.qxd 1/28/2005 12:42 PM Page 41