Sauer:ClpP purification/Untagged ClpP: Difference between revisions
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1. Suspend cells expressing ClpP in 10mL/L culture Buffer L150. | 1. Suspend cells expressing ClpP in 10mL/L culture of Buffer L150. | ||
2. Freeze at - | 2. Freeze at -80°C until use. | ||
3. Thaw by addition of 10mL/L culture Buffer L150. | 3. Thaw by addition of 10mL/L culture Buffer L150. | ||
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6. Spin at 15K rpm in a SA-600 rotor, 30’. | 6. Spin at 15K rpm in a SA-600 rotor, 30’. | ||
7. Add 30% (saturation) | 7. Add 30% (saturation) AmSO<sub>4</sub> and incubate at 4°C, 30’. | ||
8. Spin at 10K rpm, 15’; save supernatant. | 8. Spin at 10K rpm, 15’; save supernatant. | ||
9. Add to 60% (saturation) | 9. Add to 60% (saturation) AmSO<sub>4</sub> and incubate at 4°C, 30’. | ||
10. Spin at 10K rpm, 15’; save pellet. | 10. Spin at 10K rpm, 15’; save pellet. | ||
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11. Resuspend pellet in Buffer L150 (I used 5 mL per 1L culture but this was more than I needed:[[User:Marylee|Marylee]]) | 11. Resuspend pellet in Buffer L150 (I used 5 mL per 1L culture but this was more than I needed:[[User:Marylee|Marylee]]) | ||
12. Desalt into PD-10 (with fresh Buffer L150) or decrease conductivity by dilution into L150. | 12. Desalt into a PD-10 column (with fresh Buffer L150) or decrease conductivity by dilution into L150. | ||
13. HiLoad 16/10 Q Sepharose HP separation. Buffer L150 | 13. ''HiLoad 16/10 Q Sepharose HP separation.'' The mobile phase contains Buffer L150 and elution proceeds with a 200 mL linear gradient between 150mM and 400 mM KCl. | ||
14. Concentrate sample | 14. Concentrate sample by precipitation in 60% AmSO<sub>4</sub>, and then incubate at 4°C, 30’. | ||
15. Spin at 10K rpm and resuspend in Buffer L100. | 15. Spin at 10K rpm and resuspend in Buffer L100. | ||
16. HiPrep Sephacryl S-300HR column in Buffer L100. | 16. ''Gel filtration using a HiPrep Sephacryl S-300HR column'' in Buffer L100. | ||
17. Concentrate fractions on HiLoad 16/10 Q Sepharose column or by a spin concentrator. | 17. Concentrate fractions on a HiLoad 16/10 Q Sepharose column or by a spin concentrator. |
Revision as of 13:20, 10 November 2006
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 of Buffer L150.
2. Freeze at -80°C 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 4°C, 30’.
8. Spin at 10K rpm, 15’; save supernatant.
9. Add to 60% (saturation) AmSO4 and incubate at 4°C, 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:Marylee)
12. Desalt into a PD-10 column (with fresh Buffer L150) or decrease conductivity by dilution into L150.
13. HiLoad 16/10 Q Sepharose HP separation. The mobile phase contains Buffer L150 and elution proceeds with a 200 mL linear gradient between 150mM and 400 mM KCl.
14. Concentrate sample by precipitation in 60% AmSO4, and then incubate at 4°C, 30’.
15. Spin at 10K rpm and resuspend in Buffer L100.
16. Gel filtration using a HiPrep Sephacryl S-300HR column in Buffer L100.
17. Concentrate fractions on a HiLoad 16/10 Q Sepharose column or by a spin concentrator.