Berglund:MBNL

GST-MBNL Med Purification

1. Innoculate 10 mL of LB with 50 ug/mL amp (1x) and a swap from -80°C frozen BL21* cells. Incubate/shake overnight at 37°C.

2. With overnight culture, inoculate 1-3 L of LB, 50 ug/mL amp (1x). Grow cells to OD600 between 0.5 and 1.0. Can grow cells to density at 37 °C. (Uninduced sample)

3. Induce with 0.25 mM IPTG at 37°C for 2-4 hrs. (Induced sample)

4. Spin down cells at 5000 rpm, 15 mins. Drain pellet of LB media. Can freeze pellet in -20 °C.

5. Resuspend pellet in ~50 mL of Lysis Buffer, rotate at 4°C for 30 mins.

6. Incubate at 4°C for 30 mins with lysozyme (~1-2 mg/mL).

7. Sonicate 3 x 30 seconds to break up DNA clumps. Avoid aerosauling (will precipitate protein).

8. Centrifuge lysate at 15,000 rpm for 15 mins.

9. Prep GST beads. Use ~10 mL of slurry (~6 mL of settled beads). Wash 3x with up to 50 mL of lysis buffer (dont spin beads more than 3000 rpm).

10. Bind supernatant to GST beads for 0.5 -1 hour at 4 °C on rotary. (Cell Lysate sample)

11. Wash beads 3X with 50 mL of Wash Buffer A. (Wash #1 sample)

12. Wash beads 2X with 50 mL of Wash Buffer B.

13. IF CUTTING OFF GST TAG- Wash beads once with 50 mL of Elution Buffer with no reduced glutathione (to get rid of the extra salt in the wash buffer). Then resuspend beads in ~10-20 mL of cutting buffer (which is elution buffer without reduced glutathione). Add 3 aliquots precision protease and cut overnight in 4 °C on rotary. Collect protein by centrifugation (may wash beads one time with 10 mL of Elution Buffer to get more protein). (Bound Bead sample)

14. IF NOT CUTTING OFF GST TAG- Wash beads once with 50 mL of Elution Buffer with no reduced glutathione (to get rid of the extra salt in the wash buffer). Elute with fresh Elution buffer (with Glutathione) at 4 °C on rotary, with 2x 10 mL for 30 mins each, or just once with ~30 mL for 1 hr. (Cut Bead and Cut Supernatant samples)

15. Filter supernatant, through using 0.45 micron filter (with syringe), to filter out GST beads.

16. Concentrate protein supernatant to ~20 mL using Nitrogen Concentrator (30,000 MWCO for GST-MBNL, 10,000 MWCO for MBNL No GST)

17. Add ~30 mL of Q Column Buffer A’ (NO SALT) to 20 mL protein supernatant into superloop for AKTA (ensures NaCl~60mM)

18. Run protein on Q column, protein will be in flow through. Collect flow through. (Flow Through sample)

19. Concentrate flow through using Nitrogren Concentrator and appropriate MWCO for protein. Spike a little bit of salt into the flow through (300-500mM NaCl). Concentrate to a volume of 1-5 mL

20. Dialyze overnight into storage buffer

21. Aliquot and store indefinitely at -80 °C. Aliquots can be stored temporarily at -20 °C when in use.

** (Bold) indicates 100 uL samples taken for the prep

SOLUTIONS
**All BME must be freshly added to solutions

Lysis Buffer
500 mM NaCl
25 mM Tris pH 7.5
10 mM BME
5% glycerol

Wash Buffer A
1 M NaCl
25 mM Tris pH 7.5
5 mM BME

Wash Buffer B
300 mM NaCl
25 mM Tris pH 7.5
5 mM BME

Elution Buffer
10 mM reduced glutathione (FRESH)
150 mM NaCl
50 mM Tris pH 8.0 (so glutathione doesnít change pH)
5 mM BME

Storage Buffer
500 mM NaCl
25 mM Tris pH 7.5
5 mM BME
50% glycerol

Q Column Buffer A’ (No NaCl)
10 mM Tris pH 7.5
1 mM EDTA pH 7.5
5 mM BME

Q Column Buffer A
60 mM NaCl
10 mM Tris pH 7.5
1 mM EDTA pH 7.5
5 mM BME

Q Column Buffer B
1 M NaCl
10 mM Tris pH 7.5
1 mM EDTA pH 7.5
5 mM BME