Editing Taq377 Purification (section)

== '''Protocol 1'''  ==

1. Grow Rosetta BL21(DE3) pLysS transformed with pET30b-sso7d-Taq_d289 (377) in 6L LB Kan, at 37 oC shaking 180 rpm.<br> 

2. Induce with 1 mM IPTG for 3~ 4 hours at 37oC (add 1 ml 1 M IPTG to 1 L culture).<br> 

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

4. Using DI water, transfer E coli cells to 50 ml VWR tubes, spin down cells, 6000 rpm for 5 min.<br> 

5. Freeze at -20 oC (store cells at -20 oC if not use right away).<br> 

6. Add 200 ml bacterial lysis '''Buffer A''' containing 10 mM imidazole.<br> 

7. Lyse cells by thawing at room temperature.<br> 

8. Transfer cells to a beaker, homogenize gel like stuff.<br> 

9. Sonicate if it is still sticky.<br> 

10. Boil at 75 <sup>o</sup>C for 30 min, cool to 4 <sup>o</sup>C on ice.<br> 

11. Spin down pellet at 12000 rpm for 20 min at 4 <sup>o</sup>C.<br> 

12. Collect supernatant, spin again at 25000 or 30000 rpm for 20 min at 4 <sup>o</sup>C.<br> 

13. Collect cleared supernatant. Load supernatant to 10 ml Ni column.<br> 

14. Wash with '''washing buffer''' until there is no detectable protein (10 ÎŒL eluate + 75 ÎŒL Bradford reagent).<br> 

15. Wash with '''washing buffer II''' until there is no detectable protein (10 ÎŒL eluate + 75 ÎŒL Bradford reagent).<br> 

16. Elute Taq with elution '''buffer B'''. Collect 5 ml fractions. Check each fraction by SDS-PAGE.<br> 

17. Combine relatively pure fractions.&nbsp; Concentrate protein using 10 K MWCO spin column.<br> 

18. Load 5 ml Taq to HiLoad 16/60 Superdex 200 PreGrade column, equilibrated with '''GF Buffer'''.<br> 

19. Elute with GF buffer. Collect 3 ml per fraction.<br> 

20. Use an ELISA plate, check fractions containing Taq protein using 75ÎŒL Bradford reagent.<br> 

21. Take 10 ~ 15 ul to run SDS-PAGE to locate fractions containing Taq.<br> 

22. Combine fractions containing Taq, dialyse against '''Storage Buffer A''', dialyse 3 X 75 ml, 3 hour each, then 75 ml overnight.<br> 

22. Measure protein concentration. Dilute to ~ mg/ml with '''Storage Buffer A + 1/100 Storage Buffer B (ratio of A:B is 100:1).''' Use Invitrogen Taq and Qiagen Taq&nbsp; as "reference standard" to estimate specific activity. Dilute protein to 5 units/ÎŒL.<br> 

'''Buffers:''' 

Buffer A: 10 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600 mM NaCl 

Buffer B: 300 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600 mM NaCl 

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> 

GF buffer: 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 100 mM NaCl.<br> 

'''Storage Buffer A:'''<br> 

Tris-HCl, pH 8.0, 100 mM NaCl, 0.1 mM EDTA,&nbsp; 50% glycerol<br> 

<br> 

'''Storage Buffer B:'''<br> 

100 mM DTT, 10% Tween20, 10% Nonidet P-40<br> 

<br> 

'''<u>DO&nbsp;NOT&nbsp;USE the following protocols.</u>'''