1. Harvest cells and lysis cells in fresh MBP Binding buffer (20 mM Tris 7.4, 200 mM NaCl, 1 mM EDTA, 10 mM beta-mercaptoethanol, 0.5% Tween X-100). Clear the supernatant. See Expression of protein in bacterial cells for details.

2. Resuspend the amylose resin. Transfer 2-5 ml to a 15/50 ml tube.

3. Centrifuge at 700 g for 2 minutes to pellet the resin.

4. Remove supernatant. Add 10 bed volumes MBP Binding buffer (20 mM Tris 7.4, 200 mM NaCl, 1 mM EDTA, 10 mM beta-mercaptoethanol, 0.5% Tween X-100).

5. Centrifuge to pellet the resin.

6. Repeat the wash once.

7. Add cleared sample to the resin.

8. Gently agitate at 4 °C for 2 hours on a shaker.

9. Centrifuge to pellet the resin.

10. Remove supernatant.

11. Wash with 10-20 bed volumes MBP Binding buffer (20 mM Tris 7.4, 200 mM NaCl, 1 mM EDTA, 10 mM beta-mercaptoethanol, 0.5% Tween X-100).

12. Gently agitate at 4 °C for 10 minutes on a shaker.

13. Centrifuge to pellet the resin.

14. Remove supernatant.

15. Repeat the washing steps 11-14 twice.

16. Resuspend the resin in 1 bed volume MBP Binding buffer.

17. Transfer the resin to a gravity-flow column with an end-cap in place.

18. Allow the resin to settle. Remove the end-cap and allow the buffer to drain until it reaches the top of the resin bed.

19. Wash the column with 5 bed volumes of MBP Binding buffer.

20. Elute the protein with 5 bed volumes of Elution Buffer (MBP Binding buffer + 10 mM Maltose).

21. Collect 0.2-0.5 ml fractions.

22. Determine the protein fraction using a UV spectrometer, Bradford assay, or SDS-PAGE gel analysis.