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| {{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)''' | |
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| [[Buffer L:]]
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| 50 mM Tris-HCl, pH 7.6
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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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| [[Buffer L150:]]
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| Buffer L + 150 mM KCl
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| [[Buffer L400:]]
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| Buffer L + 400 mM KCl
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| [[
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| Buffer L100:]]
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| Buffer L + 100 mM KCl
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| 1. Suspend cells expressing ClpP in 10mL/L culture Buffer L150.
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| 2. Freeze at -80C until use.
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| 3. Thaw by addition of 10mL/L culture Buffer L150.
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| 4. Add 1 mg/mL lysozyme, and let sit over ice, ~1 hour.
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| 5. Sonicate lysis.
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| 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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| 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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| 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)
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| 12. Desalt into PD-10 (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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| 14. Concentrate sample with 60% AmSO4 ppt, incubate at 4C, 30’.
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| 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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| 17. Concentrate fractions on HiLoad 16/10 Q Sepharose column or by a spin concentrator.
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