Native purification of His-tagged Proteins from Sf9 cells

Native purification of His-tagged Proteins from Sf9 cells

Protocol is for 150 ml of cell culture.

1. Spin down cells and wash 1X in 10 ml ice cold PBS.

2. Resuspend pellet in 20 ml lysis buffer: 50 mM Tris pH 8.0
1 % NP-40
1 mM PMSF
1 µg/ml leupeptin

3. Transfer to oakridge tubes and sonicate at a setting of 4 for 20 seconds.

4. Spin lysate in SS-34 for 20 minutes at 10,000 rpm @ 4°C.

5. Bind supernatant to 0.5 ml Ni-NTA pre-equilibrated in lysis buffer. Rock at 4°C for 1 hour to overnight.

6. Transfer slurry to Bio-rad econo column and collect flow thru.

7. Wash with 20 ml 50 mM Tris pH 8.0, 500 mM NaCl.

8. Wash with 20 ml 50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole.

9. Elute with 5 ml 50 mM Tris pH 8.0, 500 mM NaCl, 0.5 M imidazole. Collect 1 ml fractions.

10. I generally pool fractions 1 and 2 and dialyze overnight against 2 L of desired buffer.