Sauer:ClpP purification: Difference between revisions
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'''Untagged ClpP Purification Protocol | |||
(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 20:33, 25 September 2006
Untagged ClpP Purification Protocol (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.
