Sauer:ClpP purification/Untagged ClpP
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| Line 1: | Line 1: | ||
| - | + | <font style="color:red">Buffer L:</font> | |
| - | 50 mM | + | 50 mM Tris-HCl (pH 7.6), 1 mM DTT, 0.5 mM EDTA, 10% Glycerol |
| - | 1 mM | + | |
| - | 0.5 mM | + | |
| - | 10% Glycerol | + | |
| - | + | <font style="color:red">Buffer L150:</font> | |
| - | + | ||
Buffer L + 150 mM KCl | Buffer L + 150 mM KCl | ||
| - | + | <font style="color:red">Buffer L400:</font> | |
Buffer L + 400 mM KCl | Buffer L + 400 mM KCl | ||
| - | + | ||
| - | Buffer L100: | + | <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.
