Sauer:ClpP purification/Untagged ClpP: Difference between revisions
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==Buffers== | |||
<font style="color:red">Buffer L:</font> | <font style="color:red">Buffer L:</font> | ||
50 mM Tris-HCl (pH 7.6), 1 mM DTT, 0.5 mM EDTA, 10% Glycerol | 50 mM Tris-HCl (pH 7.6), 1 mM DTT, 0.5 mM EDTA, 10% Glycerol | ||
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Buffer L + 100 mM KCl | Buffer L + 100 mM KCl | ||
==Protocol== | |||
1. Suspend cells expressing ClpP in 10mL/L culture of Buffer L150. | 1. Suspend cells expressing ClpP in 10mL/L culture of Buffer L150. | ||
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17. Concentrate fractions on a 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. | ||
==Notes== | |||
Revision as of 14:45, 15 March 2007
Buffers
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
Protocol
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.
