Bio-rad Profinity IMAC Resins User Manual download pdf (Page 26)

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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Bio-rad Profinity IMAC Resins User Manual Page 26

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