Paul.xu at 19:53, 23 October 2012

2012-10-23T19:53:27Z

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'''Day 1 '''

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

2. Add [[AutoInduce Protocol|autoinduce]] components  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. 

3. In the afternoon, add 30 ml culture to 1 L autoinduce medium. Grow at 37 oC O/N.

'''Day 2''' 

4. Spin down cells, 6000 rpm for 5 min.  Transfer to 50 ml VWR tubes using dH2O, spin down again. 

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

6. Thaw cells in 25 oC water bucket, repeat freeze-thaw one more time. 

7. Sonicate until the liquid is not sticky.

8. Spin at 12000 RPM for 15 min at 4 oC.

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

10. Spin at 25000 RPM (ultracentrifuge) for 15 min at 4 oC. 

11. Load cleared supernatant to Ni column. 

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. 

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

14. Load eluate into amylose column, collect flow through. 

15. Wash column with 1X PBS. 

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

17. Concentrate protein using a spin concentrator or dialyse against  50% glycerol in 1 XPBS 

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

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

20. Store protein at -80 oC.

21. Regenerate amylose beads: 

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.. 

 

'''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''':  Buffer A + 0.5% Triton X-100 

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

'''Buffer B''':  300 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600 mM NaCl, 0.3 mM EDTA, 10% Glycerol, 1 mM beta-ME '''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. 

[[Category:Protocols]]

MBP Fusion Protein Purification - Revision history

Paul.xu at 19:53, 23 October 2012