Editing Purification of VLP

== Medium and Buffers. ==

'''LB'''

500 g LB powder, 80 g Na<sub>2</sub>HPO<sub>4</sub>.7H<sub>2</sub>O, 10 g KH<sub>2</sub>PO4, add 20 L ddH2O, aliquote &nbsp;1 L medium into 2 L culture flask, autoclave

'''50X 5052'''

250 g glycerol, 25 g glucose, 100 g alpha lactose, add ddH<sub>2</sub>O to 800 ml, stir&nbsp;until dissolve, add ddH<sub>2</sub>O 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&nbsp;0.1% Triton X100

'''Low Salt Buffer B'''

20 mM KPO<sub>4</sub>, 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 KPO<sub>4</sub>, pH7.0 or pH 7.4,&nbsp; 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&nbsp;0.1% Triton X100

'''Inclusion Body Solubilization Buffer'''

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

== Culture and Harvest Cells ==

1, Streak a VLP clone in Nico21(DE3)pLysN&nbsp; 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&nbsp;tube with&nbsp;15 ml LB/KC medium

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

4. Pour 15 ml the above&nbsp;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&nbsp; 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&nbsp;induction overnight '''providing you culture&nbsp; 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&nbsp;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&nbsp; tube. Label as (Clone#) S

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

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

17. Load (Clone#) P solution into another&nbsp;5 ml Ni FF column at room temperature,&nbsp;&nbsp;label the Ni column as&nbsp;(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 &nbsp;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,&nbsp; &nbsp;program '''6''' on PC, elute protein directly into 2X 1 ml Q FF + 1 ml SP HP.

Buffers: '''High Salt Buffer A''',&nbsp;'''Buffer 2''',&nbsp;<span style="color:#FF0000">'''Low Salt Buffer B'''</span>

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<br/></s>

<s>24. Put the red columns where on the platform and transfer the label&nbsp; 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&nbsp;AKTA Prime Plus,&nbsp; &nbsp;program '''9''' on PC. Collect fractions.&nbsp;

Buffers: '''Buffer 2,'''&nbsp;<span style="color:#FF0000">'''Buffer B for XC'''</span>

26. Measure protein concentration&nbsp;in peak tubes&nbsp;using nanodrop&nbsp;

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

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