Editing PKA Purification

Purification of PKA 

<br>Requires: 5 L LB 

25 g LB powder. 

add 50 ml 20XP buffer to 1 L LB 

'''Day 1''' 

1. 5 PM: Innoculate 4 ml LBY/Kan/Chl with PKA in DH5alpha (clone '''pG-KJE8'''), grow at 37 oC o/n shaking.<br> 

pG-KJE8 will refold misfold preotein<br> <br> 

'''Day 2''' 

<br> 2. Make 200 ml LBY/Kan/Chl at 9 AM, add 4 ml culture, grow at 37 oC for 6~8 hours shaking. 

&nbsp;3, Make 6 L LBY/Kan/Chl, Add 1 ml 1000X Kan and 1 ml 1000X Chl to 1 L LBY, add 50 ml o/n culture to 1 L LBY/Kan/Chl. 

4. Add 1 ml of 20% arabinose to 1 L culture, induce at 25 oC or 4 oC for o/n, shaking 120 to 180 rpm. 

<br> 

'''Day 3''' 

5. Spin down cells, 6000 rpm for 5 min'''.''' <br> 

6. Transfer to 50 ml tubes using dH2O, spin down cells. 

7. Add 30 ml lysis buffer (Buffer A containing 10% glycerol and 5 mM beta-ME<strike></strike>). Freeze cells with dry ice. 

8.&nbsp; Thaw cells in 25 oC water bucket. 

9.&nbsp; If cell is not sticky, add 50 ul lysozyme, incubate at RT for 30 min or in the cold room overnight. 

10. Use sonicator to break the sticky DNA.<br> 

<br><strike></strike>11. Spin at 12000 RPM for 30 min. 

12. Collect supernatant. Do not discard pellet.

13. Spin for 30 min at 25000 RPM (ultracentrifuge), 4 oC. <br> 

14. Load cleared supernatant to Qiagen 10 ml Ni column,&nbsp; do not throw away flow through (FT). 

15. Wash beads with buffer A until no protein is detectable (using Braford reagent). 

16. Wash beads with 20 ml Wash Buffer II (Buffer A with <u>50 mM immidozol)</u>. 

17. Elute PKA with ~25 to 45 ml Buffer B, collect 2 ml fractions.&nbsp;<br> 

18. Measure protein concentration using 10 ul from each fraction and 75 ul Braford reagent.<br> 

19. Run SDS-PAGE to check purity. <br> 

20. Combine PKAalpha fractions 

21. Load to Tosho gel filtration column (FPLC). Elute protein using 20 mM Tris-HCl, pH 8.0 or 7.5, 0.1 mM EDTA, 5 mM beta-ME, 200 mM NaCl. 

22. Combine fraction containing PKAalpha. 

23. Concentrate protein using spin concentator (10 KDa). 

24. Measure protein concentration using nanodrop. <br> 

25. Aliquot 2 ml/tube, freeze in dry ice and store them at -80 oC <br> '''Buffer:''' 

Buffer A: 10 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600 mM NaCl, 10% Glycerol (optional), 0.3 mM EDTA, 2 mM beta-ME 

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 

Washing Buffer: 20 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600 mM NaCl. 

Washing Buffer II: 50 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600 mM NaCl.<br> 

[[Category:Protocols]]