6X His-tagged protein purification

Lirong Zeng, 04-08-2005 optimized on 08-01-2005

o 1. Grow bacteria (from LB or colony) in 5ml LB/Amp/Chlor 37 C to OD600 1.0 2. Transfer the 5ml culture to 250 flaks with 50ml LB/Amp/Chlor (make OD600 ~0.1) 3. Grow at 14oC overnight 250rpm 4. In the morning (OD600 ~0.6) add 100 ~ 200 uM IPTG (For E2s, use 200 uM) (50ul from 0.1M stock) 5. Incubate 8 hr 14oC 250rpm 6. Pellet cells 15 min 5000 x g 4 oC (pellet can be stored at -80oC)

The following steps on ice or 4 oC: 7. Resuspend cells in 10ml Equilibration buffer. Mix gently by inversion (break pellet) 8. Add lysozyme to 1mg/ml (200ul from 50mg/ml stock)

9. (optional) Add 1.5 ml of N-laurylsarcosine (1.5% from 10% stock in STE buffer), and glycerol to a final concentration at 10%, mix gently.

10. Sonicate on ice (5 pulses #5, 5 seconds each pulse) 11. Add Tx-100 or tween-20 to 1% (500ul from 20% stock). Mix by inversion. 12. Incubate on ice for at lest 30-60 min. Mix gentle avoiding bubbles. 13. Spin 15min at 15000 x g 4 oC 14. Filter using 2-4 layers of cheesecloth 15. Incubate cell lysate with equilibrated agarose beads (batch) end-to-end for 60 mins at 4 oC 16. Spin 500 X g 5 mins. Remove and save the supernatant for SDS-PAGE 17. Add 10X volume of Wash buffer to the affinity gel and wash end-to-end for 25 mins. Repeat this step for 2 times. Discard the washes. 18. Add 1 ~2 gel volume of Elution buffer and mix the gel gentely by shaking in the orbital shaker at 14C for 10 mins. 19. Centrifuge the mixture at 500 X g for 5 mins. Save the supernatant. 20. Repeat step 18 and 19 to elute more proteins.

Equilibration buffer: 50 mM sodium phosphate, pH 8.0 0.3 M sodium chloride 5 mM imidazole (depends on difference proteins) 10 mM 2-mercaptoethanol 2.5 mM DTT 10% glycerol protease inhibitor cocktail (Sigma P8340) (add 50 ul cocktail/10 ml lysate)

Wash buffer: 50 mM sodium phosphate, pH 8.0 0.3 M sodium chloride 50 ~ 60 mM imidazole (depends on difference proteins)

Page 1 of 3 10 mM 2-mercaptoethanol 3 ~5 mM DTT (do not exceed 5 mM, for E1, use 4 mM, for E2s, 2.5 mM) 10% glycerol protease inhibitor cocktail (Sigma P8340) (add 30 ul cocktail/10 ml lysate)

Elution buffer: 50 mM sodium phosphate pH 8.0 0.3 M sodium chloride 400 mM imidazole (depends on protein purified. For E1 and E2s, 400 mM works well)

Note: buffers or reagents that chelate metal ions should not be used with any of the buffer. Strong reducing agent also should be avoided.

For those protein of which the His tag is buried, it needs to be purified under denature condition. In denature condition, the protein must first be solubilized with 6 M guanidine hydrochloride or 8M Urea. Make sure the pH of the denature cell extract is between 7.0 and 8.0 before applying to the affinity gel. Note that any buffers contain urea must be made fresh daily.

Cleaning HIS-Select Nickel Affinity Gel for Reuse The affinity gel should be cleaned after every run toensure that it will function properly on the next use. Ifthe same crude extract is used and it has been madeusing CelLytic B, the column can usually beregenerated with just equilibration buffer. Thedetergent in CelLytic B prevents most non-specificprotein binding to the affinity gel if used as directed.This has been done over 20 times with a crude E. coliextract with no loss of binding capacity or purity of thefinal product. All steps may be performed at room temperature or at2–8 °C.

A. General Cleaning 1. Wash the affinity gel with 2 column volumes of deionized water. 2. Clean the affinity gel with 5 column volumes of 6 M guanidine HCl (Product No. G 3272), pH 7.5. The flow rate should be no more than 5 column volumes per hour. 3. Remove the guanidine HCl solution by washing with 2 to 3 column volumes of deionized water. 4. The affinity gel can now be re-equilibrated with 2 to 3 column volumes of equilibration buffer for immediate use or it can be washed with 1 to 2 column volumes of 30% ethanol and then resuspended in 30% ethanol for storage at 2–8 °C.

B. Recharging HIS-Select Nickel Affinity Gel If the HIS-Select Nickel Affinity Gel turns from a blue toa brown or gray color, the oxidation state of the nickelhas been reduced. The reduced nickel must beremoved and the affinity gel recharged using thefollowing procedure. 1. Wash the affinity gel with 2 column volumes of deionized water. 2. Clean the affinity gel with 5 column volumes of 6 M guanidine HCl (Product No. G 3272), pH 7.5. The flow rate should be no more than 5 column volumes per hour. 3. Remove the guanidine HCl solution by washing with 2 to 3 column volumes of deionized water.

Page 2 of 3 4. Wash the affinity gel with 5 column volumes of 0.1 M EDTA (Product No. ED4S), pH 7.0 to 8.0. 5. Wash the affinity gel with 2 column volumes of deionized water. 6. Recharge the column with 2 column volumes of 10 mg/ml of nickel sulfate hexahydrate (Product No. N 4882). 7. Wash the affinity gel with 2 column volumes of deionized water. 8. The affinity gel can now be re-equilibrated with 2 to 3 column volumes of equilibration buffer for immediate use or it can be washed with 1 to 2 column volumes of 30% ethanol and then resuspended in 30% ethanol for storage at 2–8 °C.

Note: The affinity gel can also be cleaned with 0.2 Macetic acid, 1 to 2% SDS, or ethanol. The ethanol canbe used up to 100%, but the concentration percentagemust be gradually increased and decreased inincrements of no more than 25% (i.e. 25, 50, 75, 100,75, 50, 25, 0) to prevent rapid volume changes of theaffinity gel.

Considerations when work with his-tagged proteins difficult to elute from the matrix: 1. Low pH to 4.0 of the elution buffer 2. Be sure to eliminate all DNA contamination. DNA will interfere with purification. 3. Include an ammonium sulfate precipitation step before load the cell lysate to the matrix. 4. Low temperature assists binding. 5. Prepare buffer very carefully. 0.2 ~ 0.5 pH variation will make difference 6. 1 mM DTT in all solution usually prevent mis-folding of target protein. 7. Sometimes protein forms dimmers or trimers. To prevent aggregation of proteins, high concentration of salt (up to 2M NaCl or 4M MgCl2) or add glycerol or EthOH. 8. For DNA-binding protein, PEI (polyethilenimin) precipitation will be good to remove DNA ( for more info, search google !).

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