Sauer:ClpP purification/Untagged ClpP: Difference between revisions
No edit summary |
|||
Line 20: | Line 20: | ||
3. Thaw by addition of 10mL/L culture Buffer L150. | 3. Thaw by addition of 10mL/L culture Buffer L150. | ||
4. Add 1 mg/mL lysozyme, and let sit | 4. Add 1 mg/mL lysozyme, and let sit in ice, ~1 hour. | ||
5. Sonicate | 5. Sonicate to reduce viscosity. (or use DNase or Benzonase, they need Mg++ and Ca++) | ||
6. Spin at 15K rpm in a SA-600 rotor, 30’. | 6. Spin at 15K rpm in a SA-600 rotor, 30’. | ||
7. Add | 7. Add AmSO<sub>4</sub> to 30% saturation (0.164 g/mL) while gently stirring on ice and incubate on ice for 30’. | ||
8. Spin at 10K rpm, 15’; save supernatant. | 8. Spin at 10K rpm, 15’; save supernatant. | ||
9. Add to | 9. Add to AmSO<sub>4</sub> to 60% saturation (0.181 g/mL) while stirring on ice and incubate on ice 30’. | ||
10. Spin at 10K rpm, 15’; save pellet. | 10. Spin at 10K rpm, 15’; save pellet. | ||
Line 47: | Line 47: | ||
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== | ==Notes== |
Revision as of 14:49, 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 in ice, ~1 hour.
5. Sonicate to reduce viscosity. (or use DNase or Benzonase, they need Mg++ and Ca++)
6. Spin at 15K rpm in a SA-600 rotor, 30’.
7. Add AmSO4 to 30% saturation (0.164 g/mL) while gently stirring on ice and incubate on ice for 30’.
8. Spin at 10K rpm, 15’; save supernatant.
9. Add to AmSO4 to 60% saturation (0.181 g/mL) while stirring on ice and incubate on ice 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.