Sauer:ClpP purification/Untagged ClpP: Difference between revisions
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==Protocol== | ==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. | ||
2. Freeze at -80°C until use. | *2. Freeze at -80°C until use. | ||
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. | *9. Increase the AmSO<sub>4</sub> to 60% saturation (0.181 g/mL additional) 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. | ||
11. Resuspend pellet in Buffer L150 (I used 5 mL per 1L culture but this was more than I needed:[[User:Marylee|Marylee]]) | *11. Resuspend pellet in Buffer L150 (I used 5 mL per 1L culture but this was more than I needed:[[User:Marylee|Marylee]]). | ||
*11a. Alternately, you can resuspend the pellet in "saltless" buffer. At 60% saturation, there is ~2.7 M AmSO<sub>4</sub>, so the remainder in the pellet volume contibutes plenty of salt. Resuspending in 20-30 pellet volumes will allow you to go directly to the anion exchange column without wasting PD-10 columns. If you do this, spin the resuspension to clear it if insoluble material before the anion exchange step. | |||
*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% AmSO<sub>4</sub> (0.361 g/mL), and then incubate on ice for 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. | |||
==Notes== | ==Notes== | ||
To Avoid sonication, you can use DNase or Benzonase to reduce viscosity. Change the lysis buffer to contain Mg++ and Ca++ if you do. After treatment, add EDTA to chelate the metal and continue with the prep. | |||
You can make a "5X" stock buffer: 125 mM HEPES-Tris, 2.5 mM EDTA, pH 7.6. Buffers are stronger and less sensitive to dilution if they are composed of paired buffers with pKas flanking the desired pH. HEPES ~7.4 and Tris ~8.1. Start with the buffer with a pK closest to your desired pH (125 mM HEPES in this case) and add Tris base until the pH is 7.6. | |||
Make a 1 M KCl solution with 1/50th vol. of the 5X buffer above and adjust the pH to 7.6. Then making your 100, 150, and 400 mM buffers is a matter of mixing. | |||
Latest revision as of 09:43, 16 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. Increase the AmSO4 to 60% saturation (0.181 g/mL additional) 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).
- 11a. Alternately, you can resuspend the pellet in "saltless" buffer. At 60% saturation, there is ~2.7 M AmSO4, so the remainder in the pellet volume contibutes plenty of salt. Resuspending in 20-30 pellet volumes will allow you to go directly to the anion exchange column without wasting PD-10 columns. If you do this, spin the resuspension to clear it if insoluble material before the anion exchange step.
- 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 (0.361 g/mL), and then incubate on ice for 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.
Notes
To Avoid sonication, you can use DNase or Benzonase to reduce viscosity. Change the lysis buffer to contain Mg++ and Ca++ if you do. After treatment, add EDTA to chelate the metal and continue with the prep.
You can make a "5X" stock buffer: 125 mM HEPES-Tris, 2.5 mM EDTA, pH 7.6. Buffers are stronger and less sensitive to dilution if they are composed of paired buffers with pKas flanking the desired pH. HEPES ~7.4 and Tris ~8.1. Start with the buffer with a pK closest to your desired pH (125 mM HEPES in this case) and add Tris base until the pH is 7.6.
Make a 1 M KCl solution with 1/50th vol. of the 5X buffer above and adjust the pH to 7.6. Then making your 100, 150, and 400 mM buffers is a matter of mixing.
