Purification of Fc Fusion Proteins

This section details the purification of Fc fusion proteins from filtered supernatant, as performed using the HPLC apparatus.

1. Wash protein A column with 60 mL dH2O at a flow rate of 3 mL/min using a peristaltic pump. Wash no more than 2 h before use (see Note 14).

2. Equilibrate protein A column with 60 mL binding buffer at a flow rate of 3 mL/min.

3. Pump the supernatant collected from the highest producing clone through the protein A column at a flow rate of 3 mL/min (discard the effluent).

4. Wash protein A column with 60 mL of 1X binding buffer at a flow rate of 3 mL/min.

5. Pump elution buffer through the column at a flow rate of 3 mL/min. Measure the eluate using the UV monitor set to an absorbance of 280 nm. Begin to collect eluate when the A280 of fractions rises to 0.3, and continue until it descends to 0.2.

6. If necessary, adjust pH of eluate to 7.2 with 1 M Tris buffer, pH 9.0.

7. Pool fractions collected. Follow manufacturer's protocol for using HiPrep 26/10 desalting column in order to change diluent to PBS.

8. Measure final Fc fusion protein concentration using an UV spectrophotometer set at 280 nm.

0 0

Post a comment