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| {{Sauer lab sidebar}} | | {{Sauer lab sidebar}} |
| [[Sauer:ClpX purification/Protocol 1|Non-His<sub>6</sub>-tagged purification, Protocol 1]] | | [[Sauer:ClpX purification/Protocol 1|Non-His<sub>6</sub>-tagged purification, Protocol 1]] |
| ==Protocol 1==
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| *This protocol has worked successfully for Jon and [[user:kathmc|Kathleen]]
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| ===Cell growth===
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| ClpX can be expressed in a variety of cell types, but greater success usually results from strains that do not have "leaky" expression. Tight control of expression can usually be obtained with a BL21 pLysS system, but others work as well.
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| 1. Grow cells to a high density (~A<sub>600</sub>=1.0) at 37 ˚C in either LB or TB media. Then, shift temperature to 25 ˚C. Shortly thereafter, induce ClpX with 0.5–1 mM IPTG for 3 hr.
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| 2. Harvest cells by centrifugation. Resuspend in 1 mL lysis buffer (see buffer recipes below) per gram of wet cell pellet. The paste can be stored at -80 ˚C until you are ready to purify ClpX.
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| ===Lysis/ammonium sulfate((NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>) precipitation===
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| 1. Thaw cells and add 10 mL lysis buffer per gram of cell pellet. Disrupt cells by either French press or gentle sonication.
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| 2. Spin at 13–15K rpm in Sorvall RC5B centrifuge with SA600 rotor for 1 hr to pellet large cell debris.
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| 3. Transfer supernatent to appropriate tubes and spin in ultracentrafuge (Baker lab) in Beckman Ti45 rotor for 1 hr at 40K rpm.
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| 4. Remove the supernatent to new tubes. Add (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> to 35% and stir at 4 ˚C for 1 hr–overnight.
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| 5. Spin at slow speed (4K in Beckman J6-HC swinging bucket rotor) to pellet (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> precipitate. If you spin too fast here, resuspension is more difficult.
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| 6. Just in case, take supernatent and add NH<sub>4</sub>SO<sub>4</sub> to 45% and stir at 4 ˚C. Spin as in step 5.
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| 7. Resuspend the (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> pellet in lysis buffer (use minimal volume so you have a reasonable load volume for the phenyl sepharose column). After allowing as much of the precipitate as possible to resolubilize (some material typically remains insoluble), spin the sample for 1 hr at 12K in Sorvall RC5B centrifuge with the SA600 rotor. Keep the supernatent.
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| 8. Run samples of lysis supernatent and pellet, and (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> supernatents and pellets on an SDS-polyacrylamide gel to make sure you haven't left all the ClpX behind.
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| ===Phenyl sepharose column===
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| 1. Add (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> to the ClpX sample until the conductivity matches the conductivity of the phenyl sepharose equilibration buffer (buffer PS-A). Obviously, too much (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> at this step will precipitate the ClpX.
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| 2. Equilibrate a HiLoad 16/10 phenyl sepharose high performance column (Amersham) with buffer PS-A, load sample, and wash column with buffer PS-A.
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| 3. Run gradient from 0–100% buffer PS-B. ClpX generally elutes half way through the gradient.
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| 4. Run a gel of the fractions and combine those that contain ClpX.
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| ===Q sepharose column===
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| 1. Buffer exchange combined phenyl sepharose fractions containing ClpX into buffer QS-A (dialyze, do '''not''' use a PD-10 column; ClpX sticks to PD-10s). Alternatively, dilute the ClpX sample to a conductivity equivalent to that of buffer QS-A.
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| *I took the dilution route and ended up with ~500 mL of sample, which I loaded onto the column using the peristaltic pump. This may or may not save you time versus an overnight dialysis step. [[User:Kathmc|Kathleen]]
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| 2. Equilibrate a HiLoad 16/10 Q sepharose high performance column (Amersham) with buffer QS-A, load sample, and wash column with buffer QS-A.
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| 3. Run gradient from 0–100% buffer QS-B. ClpX generally elutes around 300 mM KCl.
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| 4. Run gel and pool fractions that contain ClpX. Determine the concentration by UV absorbance (extinction coefficient @280 nm=84480 cm<sup>-1</sup>M<sup>-1</sup> for ClpX<sub>6</sub>). Aliquot and keep samples frozen at -80 ˚C. Some recommend only thawing a tube once, as multiple freeze/thaw cycles results in loss of activity.
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| ===Source 15Q column===
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| To concentrate the ClpX sample, run a smaller volume Source 15Q column (Amersham) with a steep gradient using the same buffers you used for the Q sepharose column. ClpX typically sticks to spin columns used for concentration, so you will lose protein if you try to concentrate your sample this way.
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| ===More columns...===
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| If you are unhappy with the purification at this point, you can try running a gel filtration column (S200, Amersham) and/or a hydroxylapatite column.
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| ===Buffers===
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| '''Lysis buffer'''
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| 50 mM Tris-HCl, pH 8.0
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| 100 mM KCl
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| 5 mM MgCl<sub>2</sub>
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| 5 mM DTT
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| 10% glycerol
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| 1 mM PMSF (make fresh!)
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| '''Bufer PS-A'''
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| 50 mM Na-phosphate buffer, pH 7.5
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| 2 mM DTT
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| 10% glycerol
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| 0.5 M (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
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| '''Buffer PS-B'''
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| Same as PS-A, but with no (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>.
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| '''Buffer QS-A'''
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| Same as lysis buffer, but with no PMSF.
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| '''Buffer QS-B'''
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| Same as QS-A, but with 1 M KCl (or you could use NaCl here).
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| ==Who to ask about this protocol== | | ==Who to ask about this protocol== |
| Greg, Andreas, [[user:kathmc|Kathleen]] | | Greg, Andreas, [[user:kathmc|Kathleen]] |
