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