Sauer:ClpP purification

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Current revision (15:27, 10 November 2006) (view source)
 
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'''Untagged ClpP Purification Protocol
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*[[Sauer:ClpP purification/Untagged ClpP|'''Untagged ClpP Purification Protocol''']] (adapted from Joshi, et al., NSMB 2004)'''
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(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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