Paul.xu at 17:51, 24 October 2012

2012-10-24T17:51:40Z

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1.  Grow BL21 cells (with Cre) in autoinduce medium/Amp or Car.

2.  At OD600 = ~ 1,  transfer culture flask to a shaker in cold room. Continue culture O/N (16 hours).

3. Spin down cells, lyse cells using freeze-thaw or lysozyme (100 ug/ml) and DNase (100 ug/ml), do not sonicate.

4. Centrifuge lysate for 30 min at 12000 RPM. Carefully transfer supernatant to a new tube (no particles or turbid stuff).

5. If you accidentally "contaminate" supernatant with some loose "pellet", filter the supernatant with 0.45 u filter.

6. Load the supernatant onto an 5 ml Ni-NTA column (pre-equilibrated with wash buffer) using a peristolic pump at a speed 0.5 ~1 ml/min.

7. Wash column with 30 ml filtered wash buffer containing 20 ~50 mM imidazole.

8. Connect the column to AKTA FPLC. Use program #_ to wash and elute proteins. Collect 2 ml/minute fractions.

9. Analyze fractions from the initial flowthrough (wash) as well as fractions from the elution that contain protein.

10. For each fraction, dilute 15 ul of sample with 3 ul of 6 X SDS sample buffer. Boil at 95 oC for 2 min and load onto a 10% SDS-PAGE gel. Run at 200 V for 35 min. Stain the gel with coomassie.

11. Estimate the amount of protein in each fraction using Bradford reagent. Pool the fractions that contain Cre. Adjust protein concentration to 2.5 mg/ml using low concentration fractions. Dialyze Cre with 2X storage buffer.

12. Measure protein concentration using Bradford reagent and OD280.

13. Add DTT (final concentration 2 mM) and glycerol (final concentration 50%) ( optional: 50 mM trehalose).

14. Dispense Cre into 1 ml (2 mg/ml) aliquots, quick freeze in a dry ice/ethanol bath and store at â80oC. 

15. Remove Endotoxin: Load purified Tat-NLS-Cre onto a MonoQ 4.6/100 PE column and elute with a linear gradient 150 mM to 1 M NaCl. Endotoxin can also be removed using an Endotrap Blue 1/1 column from Profos.

16. In vivo recombination.

Add 100 uM chloroquine to endothelia cells cultured in serum-free DME medium. Add Tat-NLS-Cre to final concentration of 150 ug/ml. Incubate for 1.5 hour. Wash with DME Culture cells with complete medium for 24 hours. 

 '''Lyse Buffer:'''  Buffer A.

'''Wash Buffer:'''  Buffer A

'''Elute Buffer: '''Buffer B (300 mM imidazole).

'''Storage Buffer: '''TBS, 2 mM DTT, 50% Glycerol.

[[Category:Protocols]]

Tat-NLS-Cre-FPLC-Purification - Revision history

Paul.xu at 17:51, 24 October 2012