Crm at 16:13, 20 July 2019

2019-07-20T16:13:33Z

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== Medium and Buffers. ==

'''LB'''

500 g LB powder, 80 g Na2HPO4.7H2O, 10 g KH2PO4, add 20 L ddH2O, aliquote  1 L medium into 2 L culture flask, autoclave

'''50X 5052'''

250 g glycerol, 25 g glucose, 100 g alpha lactose, add ddH2O to 800 ml, stir until dissolve, add ddH2O to 1 L, autoclave

'''High Salt Buffer A (the same as VLP Lysis Buffer)'''

20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM Imidazole, 0.1% NP-40 or 0.1% Triton X100

'''Low Salt Buffer B'''

20 mM KPO4, pH 7.4, 50 mM NaCl, 300 mM Imidazole

'''Buffer 2'''

20 mM Tris-HCl, pH 8.0, 50 mM NaCl.

'''Buffer B for XC'''

20 mM KPO4, pH7.0 or pH 7.4,  1.5 M NaCl

For VLP with pI >8, use pH 7.4, for pI <8, use pH 7.0

'''VLP Lysis Buffer'''

20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM Imidazole, 0.1% NP-40 or 0.1% Triton X100

'''Inclusion Body Solubilization Buffer'''

20 Tris-HCl, pH 8.0, 8 M Urea, 20 mM Imidazole, 50 mM NaCl

== Culture and Harvest Cells ==

1, Streak a VLP clone in Nico21(DE3)pLysN  onto a LB/KC plate, label the plate (write clone #), grow at 37 oC incubator O/N, agar side up

2, Wash E coli into an autoclaved glass culture tube with 15 ml LB/KC medium

3. Culture cells for 1 hour at 37 oC in a shaker incubator

4. Pour 15 ml the above starter stock into 1 L LB/KC medium, add 20 ml of 50X 5052 using autoclaved 100 ml glass graduated cylinder

5. Grow cells to OD600  0.6 ~ 1.0

6. Add 0.5 ml 1 M IPTG, induce at 37 oC for 4 to 6 hours. You can also leave the induction overnight '''providing you culture  the cells overnight at 16 oC in the cold room downstairs'''. Sometimes cells may be lysed during overnight culture. So it would be better to spin down cells after 4 hours induction.

7. Spin down cells, 5 min at 5000 rpm using a high speed centrifuge.

8. Combine cells into one centrifuge tube, use LB/KC medium (supernatant liquid from previous spin) to wash cells into one tube. Spin again to collect pellet.

9. Record wet cell weight. Label tube

10. Store cells (tube) at -80 oC freezer for 1 hour or overnight.

== Purification ==

11 .Warm up cell pellet, add 200 ml VLP Lysis Buffer.

12. Sonicate 2 min (do not use homogenizer as it is more efficient to use sonicator for VLP).

13. Spin using ultra centrifuge, 30,000 rpm, 4 oC, 30 min.

14. Combine supernatant into one 250 ml  tube. Label as (Clone#) S

15. Combine pellet into another tube using  150 ml  Inclusion Body Solubilization Buffer, label as (Clone#) P

16. Load supernatant (Clone#) S  into one 5 ml Ni FF column in fridge, label the Ni column as (Clone#) S 

17. Load (Clone#) P solution into another 5 ml Ni FF column at room temperature,  label the Ni column as (Clone#) P

18. Wash both Ni FF columns with 100 ml '''High Salt Buffer A'''.

19. Use appropriate connectors, connect Ni FF column to AKTA Prime Plus FPLC, connect 2 X 1 ml Q FF and 1 ml SP HP column to the outlet of  Solenoid valve

20. Test run using '''program 10''' on PC. Check if there is any leaks, and check computer if it is collecting data

21. Purify protein using AKTA Prime Plus,   program '''6''' on PC, elute protein directly into 2X 1 ml Q FF + 1 ml SP HP.

Buffers: '''High Salt Buffer A''', '''Buffer 2''', '''Low Salt Buffer B'''

22. Disconnect Ni FF column, <s>move 2 X 1ml Q FF + 1 ml SP HP to FPLC.</s>

23. Test run using program 10 on PC, Check if there is any leaks, and check computer if it is collecting data.

<s>24. Switch out the tube rack with a clean new one </s>

<s>24. Put the red columns where on the platform and transfer the label  from the green column to the red ones</s>

24. Check for leaks with program 10 and don't forget to switch the Buffer B to XC

25. Purify protein further using AKTA Prime Plus,   program '''9''' on PC. Collect fractions. 

Buffers: '''Buffer 2,''' '''Buffer B for XC'''

26. Measure protein concentration in peak tubes using nanodrop 

27. Take 30 ul solution + 10 ul 4X SDS-PAGE Loading Buffer, boil 10 min, and run SDS-PAGE gel to check purity.

28. Label and store tubes with protein in 4 oC fridge.
[[Category:Protocols]]

Purification of VLP - Revision history

Crm at 16:13, 20 July 2019