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Star Republic: Guide for Biologists

Purification of GST-fusion protein

1. Harvest and lysis cells in 1X Extraction buffer (PBS/0.5%Tween X-100). Clear the supernatant. See Expression of protein in bacterial cells for details.

2. Resuspend the glutathione resin. Transfer 2-5 ml to a 15 or 50 ml tube.

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

4. Remove supernatant. Add 10 bed volumes Extraction/Wash buffer (PBS/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 1X Extraction/Wash buffer (PBS/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 1X Extraction/Wash 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 1X Extraction/Wash buffer.

20. Elute the protein with 5 bed volumes of Elution Buffer (100 mM Tris (pH 8.5-9.0), 20 mM reduced glutathione).

21. Collect 0.2-0.5 ml fractions.

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