Editing MBP Fusion Protein Purification

'''Day 1 ''' 

1. 9 AM: Innoculate 200 ml LBY/Kan/Chl with N-6XHis-MBP fusion in BL21 (DE3) pLysS, grow at 37 <sup>o</sup>C shaking. 

2. Add [[AutoInduce Protocol|autoinduce]] components&nbsp; to 6L LBY Medium. ''i.e.,'' add '''50 ml''' 20xP Buffer, '''20 ml''' of 50 x 5052, '''2 ml''' 1M MgCl2, '''1.2 ml '''1000X Kan, '''1.2 ml''' 1000X Chl to 1 L LBY medium.<br> 

3. In the afternoon, add 30 ml culture to 1 L autoinduce medium. Grow at 37 <sup>o</sup>C O/N. 

'''Day 2'''<br> 

4. Spin down cells, 6000 rpm for 5 min.&nbsp; Transfer to 50 ml VWR tubes using dH<sub>2</sub>O, spin down again.<br> 

5. Add 30 ml Lysis Buffer. Freeze cells for 30 min with dry ice. 

6. Thaw cells in 25 <sup>o</sup>C water bucket, repeat freeze-thaw one more time.<br> 

7. Sonicate until the liquid is not sticky. 

8. Spin at 12000 RPM for 15 min at 4 <sup>o</sup>C. 

9. Collect supernatant into ultracentrifuge tubes, balance to 0.1 g.<br> 

10. Spin at 25000 RPM (ultracentrifuge) for 15 min at 4 <sup>o</sup>C.<br> 

11. Load cleared supernatant to Ni column. <br> 

12. Wash column with '''Washing Buffer''' containing '''10 or 20 mM''' (not more than 20 mM) Imidozale. Take 10 ul from each 1 ml fraction and check protein with Bradford reagent, wash until no protein is detectable. <br> 

13. Elute protein with '''Buffer B''' (containing 300 mM&nbsp; Imidozale), use Bradford reagent to check protein in fractions. Elute unitl there is no protein coming out from column.<br> 

14. Load eluate into amylose column, collect flow through.<br> 

15. Wash column with 1X PBS.<br> 

16. Elute 6XHis-MBP fusion protein using '''M-PBS Buffer'''.<br> 

17. Concentrate protein using a spin concentrator or dialyse against&nbsp; 50% glycerol in 1 XPBS <br> 

18. Measure protein concentration and run SDS-PAGE to check purity.<br> 

19. If protein is an enzyme and was concentrated by spin concentrator, add 80% Glycerol to final concentration of 20%.<br> 

20. Store protein at -80 <sup>o</sup>C. 

21. Regenerate amylose beads:<br>

Water 3 - column volumes, 0.1% SDS - 3 column volumes, Water - 1 column volume, Column Buffer - 5 column volumes or soak in 20% ethanol for long term storage..<br>

<br> 

'''Buffers:''' 

'''Buffer A''': 20 mM Tris-HCl pH 8.0, 600 mM NaCl, 10 mM Imidozale, 10% Glycerol (optional), 0.3 mM EDTA, 1 mM beta-ME 

'''Lysis Buffer''':&nbsp; Buffer A + 0.5% Triton X-100<br> 

'''Washing Buffer''': 20 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600 mM NaCl,&nbsp; 10% Glycerol (optional) 

'''Buffer B''':&nbsp; 300 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600 mM NaCl, 0.3 mM EDTA, 10% Glycerol, 1 mM beta-ME<br> '''M-PBS Buffer''': 10 mM maltose, 1XPBS, 10% Glycerol (optional) 

'''LBY Medium:''' 25 g LB powder, 5 g yeast, tap water to 1L, autoclave 30 min.<br><br> 

[[Category:Protocols]]