Difference between revisions of "Sauer:ClpP purification"

From OpenWetWare
Jump to: navigation, search
(2 intermediate revisions by one other user not shown)
Line 1: Line 1:
{{Sauer lab sidebar}}
{{Sauer lab sidebar}}
'''Untagged ClpP Purification Protocol
*[[Sauer:ClpP purification/Untagged ClpP|'''Untagged ClpP Purification Protocol''']] (adapted from 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.

Latest revision as of 11:27, 10 November 2006


Home        Protocols        Lab Members        Materials        Equipment        Links        Internal