Sauer:ClpP purification/Untagged ClpP

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[[Buffer L:]]
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<font style="color:red">Buffer L:</font>
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50 mM Tris-HCl, pH 7.6
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50 mM Tris-HCl (pH 7.6), 1 mM DTT, 0.5 mM EDTA, 10% Glycerol
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1 mM DTT
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0.5 mM EDTA
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10% Glycerol
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<font style="color:red">Buffer L150:</font>
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[[Buffer L150:]]
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Buffer L + 150 mM KCl
Buffer L + 150 mM KCl
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[[Buffer L400:]]
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<font style="color:red">Buffer L400:</font>
Buffer L + 400 mM KCl
Buffer L + 400 mM KCl
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[[
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Buffer L100:]]
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<font style="color:red">Buffer L100:</font>
Buffer L + 100 mM KCl
Buffer L + 100 mM KCl

Revision as of 20:27, 11 October 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 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.

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