Bio-Rad Profinity IMAC Resins - Manual (Page 26)

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The binding capacity of the Profinity ™ IMAC resin is ~15 mg His-tagged protein per
ml resin (please refer to Table 1 for determination of dynamic binding capacity
conditions). Larger amounts of protein will require use of a larger column.
Additional Materials
• Chromatography system (such as the Bio-Rad BioLogic DuoFlow ™
system)
• Equipment for determining total protein concentration within the lysate.
Method
1. Equilibrate the column with at least 5 column volumes of binding buffer.
2. Add or dilute sample in binding buffer and load onto the column using a
desired flow rate.
The choice of binding buffer will vary based on the properties of the sample to
be purified. Sodium or potassium phosphate are recommended as general
starting buffers; for example, 50 mM sodium phosphate, 300 mM NaCl, 5 mM
imidazole, pH 8.0. Binding of His-tagged protein on the Profinity IMAC resin is
optimal in the pH range of 7–8.
The column may be run at flow rates up to 600 cm/hr. Higher binding of
His-tagged proteins will be achieved at lower flow rates. Average binding
capacities of the Profinity IMAC resin range between 10 and 15 mg
His-tagged protein/ml resin.
3. Collect fractions.
These fractions represent unbound proteins.
4. Wash the resin with at least 5 column volumes of wash buffer to remove
unbound sample.
Wash out remaining unbound solutes. Repeat wash steps as necessary for
the A
280
to be at or near the baseline.
5. Collect fractions from wash steps.
Pool recovered unbound proteins with fractions collected in step 3.
6. Elute bound proteins with 5 column volumes of elution buffer. Collect
1 ml fractions.
The choice of elution buffer will vary depending on the procedure used. For
example, a range of imidazole (100–500 mM) may be used to elute bound
protein from the Profinity IMAC resin.
7. Repeat elution steps 2 to 4 more times.
Save the eluates for further analysis; for example, A
280
, SDS-PAGE,
ELISA, etc.
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