Editing Purification of VLP (section)

== 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]]