Bio-Rad Profinity IMAC Resins - Manual (Page 33)

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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
Part 1: Protein Binding
1. Purge the instrument with binding buffer and then elution buffer.
2. Equilibrate the column with 5 column volumes of binding buffer (0% B) at
1 ml/min (or according to the manufacturer’s instructions).
3. Begin collecting 1 ml fractions.
4. Load lysate at desired flow rate.
To increase yield, the lysate can be loaded at lower flow rates.
Note: Monitor the backpressure, which will increase during sample
application.
5. Wash the column with 10 column volumes of wash buffer (0% B) to elute
unbound sample material.
Note: If the target protein will be eluted, continue onto Part 2. If the protein
will be renatured on-column, skip to Part 3.
Part 2: Eluting the Denatured Target
1. Elute the bound sample with 5–10 column volumes of elution buffer at
desired flow rate.
2. Equilibrate the column with 10 column volumes of binding buffer. Stop
collecting fractions.
3. Assess protein purity and recovery of fractions containing His-tagged
protein.
Use an activity assay (Bio-Rad’s protein assay kit), SDS-PAGE, or western blot
analysis with anti-histidine antibodies or antibodies specific to the target
protein.
Part 3: Purification and On-Column Renaturation of Proteins
1. Wash the column (containing the bound protein) with 10 column volumes
of urea binding buffer.
2. Apply a linear gradient from 100% urea-binding buffer to 100% refolding
buffer over 60 min at 0.5 ml/min. Refolding is initiated by a descending
gradient from 8 to 0 M urea.
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