Editing His-Tat-NLS-Cre-Purification (section)

== Protocol 5 ==

Large scale (6L) purification of TAT-Cre (by Jing Wang)

<br/>Competent cells: E.coli Tuner (DE3) pLacI (Novagen)<br/>Plasmid: pTriEx (Novagen)-His-TAT-NLS-Cre



PLacI (Novagen) contaning pTriEx -His-TAT-NLS-Cre (Novagen)

1. Inoculate 150ml LB medium. (50ug/mg Carbenicillin---sigma and 34 ug/ml chloramphenicol---sigma in 500ml flask and grow O/N at 37 oC.

<br/>2. Remove 1ml LB for OD600 blank. Add 40ml culture to 1L pre-warmed LB (50ug/ml Carbenicillin and 34ug/ml Chloramphenicol) in 2L flasks. Grow at 37C until OD600 is <0.600.

<br/>3. Aliquot 100 ml for control.

<br/>4. Add 0.5 mM IPTG (2ml, 0.5M stock to each 4L flask) and grow for another 3-4 hrs. Check OD again if possible.

<br/>5. Cool down on ice for 10min and harvest in 1L conical flasks and spin 6000 RPM, 4oC for 10 min, aspirate supernatant completely and freeze pellets on dry ice/ethanol both, store at -80C.



'''Bacterial Lysis'''

1. Prepare lysis buffer 100ml, take the frozen bacterial on ice and add 10ml to each 1L flask. Wait a few min on ice and resuspend well bacterial pellets, wash with 5ml the same buffer. Transfer to new tubes. Gently mix and incubate 15 min at RT.

2. Add Bugbuster detergent (Novagen#70921), gently mix for 15min RT.

3. Transfer to centrifuge tubes and spin down at 16,000rpm, 4C, 25min.

4. Pool all cleared lysate and filter through 0.22um.<br/>Save 100ul for SDS-PAGE analysis.

<br/>'''Nickel Column'''

1. Wash Ni-NTA slurry (Qiagen) twice with bead wash buffer (prep. 100ml). resuspend in 1 Vol. bead wash buffer.

2. Save 4ml slurry for pre-loading (2ml/ea.).

3. Divide left-over slurry to 5---50ml tubes. Spin (500rpm, 1min) and aspirate bead wash buffer.

4. Calculate cleared lysate evenly. 1ml packed bead/ 6ml cleared lysate and add to the slurry.

5. Mix gently shaking at 4C at least 1-2hr.

6. Load onto 2 plastic columns (Bio-rad Econo-columns, cat.#737-1512). Preload with the saved 2m l each bead slurry.

7. Let lysate flow through. Save 100ul for SDS-PAGE.

8. Wash column with >10 vol. column wash buffer, O/N Is OK

9. Elute column with elution buffer (100ml), collect 2ml fraction each in cold room.

10. Determine pro-con. By Bradford Assay and pool fractions.



'''Dialysis'''

1. Dialyze (10K MWCO, Slidelizer, Perirce) against 100 vol. dialysis buffer >2hrs, repeat 1X or O/N.

<br/>2. Dialyze against 4L glycerol buffer to concentrate. Filter through 0.22 mm filter.

Aliquot in hood and slow freeze (-20 C O/N, transfer to -80C).

<br/>'''Clean column'''

1. wash w/ 2 vol. 6M GuHCl, 0.2 M acetic acid.<br/>2. wash w/ 10 vol dH2O.<br/>3. wash w/ 3 vol 2% SDS.<br/>4. wash w/ 1 vol. 25% EtOH.<br/>5. wash w/ 1 vol. 50% EtOH.<br/>6. wash w/ 1 vol. 75% EtOH.<br/>7. wash w/ 1 vol. 100% EtOH.<br/>8. wash w/ 1 vol. 75% EtOH.<br/>9. wash w/ 1 vol. 50% EtOH.<br/>10. wash w/ 1 vol. 25% EtOH.<br/>11. wash w/ 5 vol. dH2O.<br/>12. wash w/ 5 vol. 0.1MEDTA, pH8.0<br/>13. wash w/ 10 vol dH2O.<br/>14. recharge with 5 vol. 0.1M NiSO4.<br/>15. wash w/ 10 vol dH2O.<br/>transfer to tubes, 4C store.

'''Solutions:'''

<br/>'''Stock 1'''<br/>10X phosphate buffer: 0.5 M NaHPO4 70.89g<br/>50mM Tris 6.06g<br/>adjust pH to 7.8, filter 2L

'''Stock 2 '''<br/>5M NaCl

'''Stock 3 '''<br/>1M imidazole adjust pH 7.0, filter

'''Lysis buffer''' 100ml<br/><br/>'''10X phosphate buffer''' 10ml (stock1)<br/>0.5M NaCl 10ml (stock2)<br/>10 mM imidazole 1ml<br/>1X protease inhibitors (EDTA-free tablets, Roche) 10 tablets<br/>2 mM PMSF<br/>1mg lysozyme/ml 100mg<br/>Benzonase (1:500)<br/>79ml dH2O

Bead wash buffer column wash buffer column elution buffer<br/>(100ml) (1L) (100ml)<br/>Stock1 10ml 100ml 10ml<br/>Stock2 10ml 100ml 10ml<br/>Stock3 1.5ml 20ml 25ml<br/>dH2O 78.5ml 780ml 55ml

phosphate 1x buffer<br/>NaCl 0.5M 0.5M 0.5M<br/>Imidazole 15mM 20mM 250mM

<br/>'''Dialysis buffer:'''<br/>0.6M NaCl<br/>20 mM HEPES<br/>pH 7.4

'''Glycerol buffer: '''<br/>50% Glycerol<br/>0.6M NaCl<br/>20 mM HEPES<br/>pH 7.4<br/>Notes:<br/>1. keep cold as normal protein purification.<br/>2. wear gloves, TAT-Cre can transducer your cells too.