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

== Current Working Protocol ==

<u>'''Lysis Buffer''':</u>&nbsp; 25 mM&nbsp; &nbsp;KPO4 , pH 7.5, 500 mM NaCl, 0.1% NP-40, 10 mM imidazole, 10% glycerol,&nbsp; 1 mM beta-ME.

<u>'''Buffer A'''</u>,&nbsp; &nbsp;25 mM&nbsp;KPO4 , pH 7.5,&nbsp; 500 mM NaCl,&nbsp;'''10 mM&nbsp;'''imidazole,&nbsp;10% glycerol.&nbsp; Store at 4 oC&nbsp; .



'''<u>Ni Washing buffer</u>:'''&nbsp;the same as Buffer A. Store at 4 oC&nbsp;.<br/>'''<u>Ni Elution buffer</u>''':&nbsp; 50 mM K Phosphate Buffer,&nbsp;'''pH 7.5''',&nbsp;'''200 mM NaCl''',&nbsp;&nbsp; &nbsp;'''150 mM imidazole''',&nbsp; 10% glycerol. Store at 4&nbsp;<sup>o</sup>C or in cold room.

'''<u>Ni Elution buffer II</u>''':&nbsp; 50 mM K Phosphate Buffer,&nbsp;'''pH 7.5''',&nbsp;'''100 mM Arginine or Histidine''',&nbsp;&nbsp; &nbsp;'''150 mM imidazole''',&nbsp; 10% glycerol. Store at 4&nbsp;<sup>o</sup>C or in cold room.---Need to test

'''<u>Cre Dilution Buffer:</u>'''&nbsp;50 mM K Phosphate Buffer,&nbsp; pH 7.5,&nbsp; '''100 mM arginine,&nbsp;'''10% glycerol.&nbsp;&nbsp;Store at 4 oC.

'''<u>SP Washing Buffer:</u>'''&nbsp;50 mM K Phosphate Buffer,&nbsp; pH 7.5,&nbsp; 10% glycerol.&nbsp;&nbsp;Store at 4 oC .

'''<u>SP Elution Buffer</u>''': 50 mM Phosphate Buffer, pH 7.5&nbsp;,&nbsp;'''1.5 M'''&nbsp;NaCl,&nbsp; 100 mM arginine, 10% glycerol.&nbsp;&nbsp;check pH to see if the buffer pH is ~8.0. Store at 4 oC .

'''<u>Dialysis Buffer</u>''': '''50% glycerol,''' 500 mM NaCl, 20 mM&nbsp;'''HEPES''',&nbsp; pH 7.4, '''100 mM Arginine''',&nbsp;'''0.1 mM EDTA'''.&nbsp; &nbsp;'''1 mM'''&nbsp; DTT, Store at 4 oC or in cold room.

'''<u>1 M Arginine:</u>'''&nbsp; 174.2g in 1L ddH2O. FIlter and store at 4 oC. Aliqute to 50 ml and store at -20 oC for long term storage. Store at 4 oC or in cold room. Arginine is very expensive.

1. Transform&nbsp; BL21 star (DE3) with pET28b-Cre (Clone #&nbsp;374), plate on LB/Kan, grow on 37 oC o/n.

2, Pick a few well isolated colonies, grow in 200 ml&nbsp;LB/Kan with shaking overnight at 37 oC. &nbsp;Make Glycerol stock.&nbsp;

3. Make 20 L&nbsp; LB-G&nbsp;medium



310 g 2xYT&nbsp;&nbsp;powder

150g NaCl

40 g Peptone or tryptone

200 ml glycerol.

autoclave, use 8 L&nbsp; for each purification.



4. Aliquote&nbsp;&nbsp;the overnight culture in a ratio of 1:50 to 1 L&nbsp; LB-G&nbsp;medium and incubate with vigorous shaking at 37 oC until an OD600 >&nbsp;1, usually 2~4 hours

5. Induce Cre expression by adding 1 M IPTG solution to a final concentration of&nbsp; &nbsp;1&nbsp; mM (add&nbsp; 1 ml of 1 M IPTG to 1 L) and induce&nbsp; &nbsp;2~4 hours at 37 oC, or 0.2 mM IPTG&nbsp;o/n at 25~ 30 oC.

&nbsp;6. Spin down cells,&nbsp; 6 min at 6000 rpm, RT., transfer pellet to one centrifuge tube. Weigh wet pellet, record weigh onto protein database.&nbsp;Add 100 ml Lysis Buffer to pellet and store at -80 oC o/n.

7. Thaw cell pellet, add ~400 ml&nbsp;'''cold Lysis Buffer.'''

'''If cells are not lysed, do step 8 and 9.'''

8. add&nbsp; freshly prepared lysozyme to final conc 1 mg/mL,&nbsp; and DNAse I (100 μg/mL) or benzonase (final conc 20 U/ml).

9.&nbsp; Incubate for&nbsp;&nbsp;30 ~90 min with stiring at room temperature, or cold room (4 oC) overnight, until the cells are lysed, and DNA is digested

10&nbsp;&nbsp;<u>Spin again at 25,000 rpm for 35 min at 4 oC on ultracentrifuge.</u>

<u>If vaccum can not go down, or speed can not go up to > 3000 rpm</u>

*<u>stop centrifuge.</u>
*<u>Check if centrifuge tube is broken or missing a seal ring.</u>
*<u>Check if centrifuge caps are tighten</u>

Note:

the vaccum pump could be damaged by evaporated salt and water if centrifuge tube is leaking.

Rotor should be&nbsp; cooled down using dry ice. Remove residual water with clean paper or cloth .

11. Resuspend pellet with 200 ml of Lysis Buffer. homogenize for 10 sec and spin at 30000 rpm again.

12. Combine supernatant and&nbsp; load to a 50 -100 ml Ni&nbsp; column.

13. Wash&nbsp; column with 200~ 1000 ml '''Lysis Buffer&nbsp;'''until OD280 drops to baseline.

Wash column with 2 bed vol of 1.5 M NaCl, 50 mM K-PO4, pH 7.5&nbsp;

14. Wash column with 1 CV of&nbsp;&nbsp;'''Buffer A'''.

15. Elute protein with&nbsp; 200 ml or more of&nbsp;&nbsp;'''Ni Elution Buffer. (350 ml)'''

'''16. Dilute eluted protein solution with 3 volume of&nbsp;'''&nbsp;'''<u>Cre Dilution Buffer (1000 ml). </u><span style="color:#FF0000;">Watch solution carefully, if protein is aggregating (protein solution turns to turbid), add solid arginine and stire immediately until the solution turns to clear!&nbsp;</span>'''

'''17. Load to a&nbsp;'''10 ml&nbsp;'''Q FF&nbsp;'''column (Pharmacia) connected to a column packed with 30~50 ml '''Capto&nbsp; SP ImpRes''' or&nbsp;&nbsp;'''SP FF or SP HP or 20 ml ''', preequilibrated with'''&nbsp;<u>Cre Dilution Buffer</u>. Q FF is required for removing endotoxin.'''

Dilute eluant with > 2&nbsp;volume of&nbsp;SP Dilution Buffer, load to&nbsp;Q FF + SP FF columns using P1 pump + air bubble detector.

18. Wash&nbsp;'''Q FF&nbsp;+ SP FF'''&nbsp;columns with&nbsp;'''<u>Cre Dilution Buffer</u>'''&nbsp;or '''<u>SP Washing Buffer&nbsp;</u>'''until OD280 reaches baseline, optionally , use FPLC to monitor washing.

'''optional:&nbsp;'''Disconnect Q FF.

19. Elute with 5~10 CV of&nbsp;&nbsp;'''SP Elution Buffer&nbsp;&nbsp;'''''by reverse flow (invert column)''''',&nbsp;'''collect 5 ml fractions,&nbsp;

optionally, elute using linear gradient from&nbsp;&nbsp;'''<u>Cre Dilution Buffer</u> to&nbsp;SP Elution Buffer, 10 CV'''

Cre will be eluted at ~ 0.8 M to 1.2 M NaCl at pH 7.5 ~8.0.



20. &nbsp; Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml).&nbsp;

Combine fractions containing high protein concentration

21. Wash a dialysis tube thoroughly with 0.1 N NaOH then ddH2O,&nbsp; Check if the dialysis tube is leaking.





22. Transfer Cre protein solution to a dialysis tube. Wash residual protein from tubes using 0.25 M arginine and transfer to the dialysis tube. Adjust to&nbsp;'''2 mM DTT'''&nbsp;concentration from 2 M DTT (in a box labeled as AMP-DTT,&nbsp; -20 oC fridge. For example, add 5 ul of 2 M DTT to 10 ml protein solution ( i.e., add 1:1000 dilution). Dialyse protein with&nbsp; 45~50 ml of&nbsp;'''<u>Dialysis Buffer&nbsp;</u>&nbsp;'''(in 100 ml beaker) in fridge,&nbsp;'''<u>mix occasionally for several times,</u>'''&nbsp;then dialyse&nbsp;overnight to concentrate protein, Measure OD280.&nbsp;

For freeze drying: use&nbsp;<u>Dialysis Buffer A, otherwise use Dialysis B</u>





23. Clean Q FF and SP FF columns with 5 CV of 1.5 M NaCl + 0.1 M NaOH, wash with 10 CV of water (for longterm storage, wash with&nbsp;20% ethanol).

24. Next morning, change&nbsp;'''<u>Dialysis Buffer,&nbsp;</u>'''dialyse&nbsp;for additional 1~6 hours, mix occasionally&nbsp;

25. After dialysis, transfer protein to a clean 50 ml tube. Wash dialysis tube with 3&nbsp;X 1 ml of&nbsp;'''1 M'''&nbsp;arginine, combine proteins.



26. Determine protein concentration using a nanodrop (OD280) or spectrophotometer. If concentration is too high for nanodrop, dilute 2 ul protein solution with water.&nbsp;&nbsp;&nbsp;'''Sterilize protein by filtration (0.22-um low protein binding syringe filter) inside the tissue culture hood. Aliquot to 1.5 ml tube at 1~1.2&nbsp; mg/vial.'''&nbsp;&nbsp; The glycerol stock should have a concentration of 200–450 uM Cre. Add 5, 10, 20, 30, 50&nbsp;'''ug'''&nbsp;(not ul) protein to each well (in 12 well plate) of 293T cells stably expressing loxP-sfGFP-loxP-TagRFP (clone #1053). Use&nbsp;'''Dialysis Buffer'''&nbsp;as control to test buffer toxicity.



27. Put vials immediately on ice. Create a new lot number in the protein and product databases, make sure to record all product information including protein name, protein concentration, plasmid name, clone name, competent cells, induction reagent (IPTG, autoinduce, L-arabinose), induction temperature and time, inducer concentration, weight of cell pellet, purification method, storage buffer,&nbsp; number of vials, enzyme activity, endotoxin level, purification date, assay date and URL, etc.





38. Place the freezer box in a freezer box,&nbsp; &nbsp;&nbsp;store the freezer box in -80&nbsp;<sup>o</sup>C freezer.&nbsp;&nbsp;

39. Wash Dialysis tube with water, store in&nbsp; 20% ethanol in a bottle container



'''Note: Do not use trehalose as it will promote protein aggregation. Use 1 M Arginine.'''