Difference between revisions of "Sauer:ClpP purification"

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[[Sauer:ClpP purification/Untagged ClpP|'''Untagged ClpP Purification Protocol''']]
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*[[Sauer:ClpP purification/Untagged ClpP|'''Untagged ClpP Purification Protocol''']] (adapted Joshi, et al., NSMB 2004)'''
(adapted Joshi, et al., NSMB 2004)'''
 
 
 
 
 
 
[[Buffer L:]]
 
50 mM Tris-HCl, pH 7.6
 
1 mM DTT
 
0.5 mM EDTA
 
10% Glycerol
 
 
 
 
 
[[Buffer L150:]]
 
Buffer L + 150 mM KCl
 
 
 
[[Buffer L400:]]
 
Buffer L + 400 mM KCl
 
[[
 
Buffer L100:]]
 
Buffer L + 100 mM KCl
 
 
 
 
 
 
1. Suspend cells expressing ClpP in 10mL/L culture Buffer L150.
 
 
 
2. Freeze at -80C until use.
 
 
 
3. Thaw by addition of 10mL/L culture Buffer L150.
 
 
 
4. Add 1 mg/mL lysozyme, and let sit over ice, ~1 hour.
 
 
 
5. Sonicate lysis.
 
 
 
6. Spin at 15K rpm in a SA-600 rotor, 30’.
 
 
 
7. Add 30% (saturation) AmSO4 and incubate at 4C, 30’.
 
 
 
8. Spin at 10K rpm, 15’; save supernatant.
 
 
 
9. Add to 60% (saturation) AmSO4 and incubate at 4C, 30’.
 
 
 
10. Spin at 10K rpm, 15’; save pellet.
 
 
 
11. Resuspend pellet in Buffer L150 (I used 5 mL per 1L culture but this was more than I needed)
 
 
 
12. Desalt into PD-10 (with fresh Buffer L150) or decrease conductivity by dilution into L150.
 
 
 
13. HiLoad 16/10 Q Sepharose HP separation.  Buffer L150, elute with 200 mL linear gradient between 150mM and 400 mM KCl.
 
 
 
14. Concentrate sample with 60% AmSO4 ppt, incubate at 4C, 30’.
 
 
 
15. Spin at 10K rpm and resuspend in Buffer L100.
 
 
 
16. HiPrep Sephacryl S-300HR column in Buffer L100.
 
 
 
17. Concentrate fractions on HiLoad 16/10 Q Sepharose column or by a spin concentrator.
 

Revision as of 17:24, 11 October 2006

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