Sauer:ClpP purification/Untagged ClpP

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1. Suspend cells expressing ClpP in 10mL/L culture Buffer L150.
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1. Suspend cells expressing ClpP in 10mL/L culture of Buffer L150.
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2. Freeze at -80C until use.
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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’.
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7. Add 30% (saturation) AmSO4 and incubate at 4C, 30’.
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7. Add 30% (saturation) AmSO<sub>4</sub> and incubate at 4&deg;C, 30’.
8. Spin at 10K rpm, 15’; save supernatant.
8. Spin at 10K rpm, 15’; save supernatant.
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9. Add to 60% (saturation) AmSO4 and incubate at 4C, 30’.
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9. Add to 60% (saturation) AmSO<sub>4</sub> and incubate at 4&deg;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]])  
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12. Desalt into PD-10 (with fresh Buffer L150) or decrease conductivity by dilution into L150.
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12. Desalt into a PD-10 column (with fresh Buffer L150) or decrease conductivity by dilution into L150.
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13. HiLoad 16/10 Q Sepharose HP separation.  Buffer L150, elute with 200 mL linear gradient between 150mM and 400 mM KCl.
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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.
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14. Concentrate sample with 60% AmSO4 ppt, incubate at 4C, 30’.
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14. Concentrate sample by precipitation in 60% AmSO<sub>4</sub>, and then incubate at 4&deg;C, 30’.
15. Spin at 10K rpm and resuspend in Buffer L100.
15. Spin at 10K rpm and resuspend in Buffer L100.
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16. HiPrep Sephacryl S-300HR column in Buffer L100.
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16. ''Gel filtration using a HiPrep Sephacryl S-300HR column'' in Buffer L100.
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17. Concentrate fractions on HiLoad 16/10 Q Sepharose column or by a spin concentrator.
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17. Concentrate fractions on a HiLoad 16/10 Q Sepharose column or by a spin concentrator.

Revision as of 15:20, 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.

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