Paul.xu at 13:45, 24 October 2012

2012-10-24T13:45:04Z

New page

Purification of PKA

 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. 

pG-KJE8 will refold misfold preotein 

'''Day 2'''

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

 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.

 

'''Day 3'''

5. Spin down cells, 6000 rpm for 5 min'''.''' 

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.  Thaw cells in 25 oC water bucket.

9.  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. 

 <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. 

14. Load cleared supernatant to Qiagen 10 ml Ni column,  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 50 mM immidozol).

17. Elute PKA with ~25 to 45 ml Buffer B, collect 2 ml fractions.  

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

19. Run SDS-PAGE to check purity. 

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. 

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

[[Category:Protocols]]

PKA Purification - Revision history

Paul.xu at 13:45, 24 October 2012