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

== '''Protocol 2''', ==

Based on [http://www.jove.com/video/1627/engineering-cell-permeable-protein http://www.jove.com/video/1627/engineering-cell-permeable-protein]

1. Inoculate LB/0.5% glucose [g/v]/50 μg/mL carbenicillin with HTNCre glycerol stock using a pipette tip.

2. Grow bacteria at 37°C O/N (for 16 hours).

3. Next day use the densely grown over-night culture to inoculate TB medium with 0.5% glucose, 100 μg/ml Amp (prewarmed to 37 oC) at a ratio of 1:40 or 1:50.

4. Grow bacteria in an incubator at 37°C to OD600 of 1.5.

5. Add 0.5 mM IPTG to induce for 1 h.

6. Spin down bacteria at 5000 rpm for 10 minutes.

7. Store bacteria at -20 oC.

8. Resuspend frozen bacteria pellets in 10 mL lysis buffer per liter flask culture for 15 minutes at room temperature.

9. Add 1 mg/mL lysozyme, incubate at room temperature for 15 minutes while mixing.

10. Add benzonase to 25 U/mL (1:1000) and incubated while mixing for 15 minutes at room temperature.

11. Sonicate on ice for 1.5 min with 0.5 s pulses at 45% of the power.

12. Add 1 mL cold tartaric salt buffer (TSB) per mL suspension while mixing and incubate for 5 min on ice.

13. Take 10 ul lysate for SDS-PAGE analysis (L).

14. Spin at 4°C for 30 min at 12000 RPM. Take 10 ul of supernatant (S) for SDS-PAGE analysis.

15. Transfer supernatnant to fresh 50 mL falcon tubes, add 2 mL of 50% Ni-NTA slurry per liter of initial expression culture, mix gently for 1 h at 4°C in cold room.

16. Pour the suspension into a gravity flow EconoPac column.

17. Take 10 ul flow through for SDS-PAGE analysis (FT).

18. Wash beads twice with 5 bed-volumes of washing buffer. Take 10 ul from washing fractions for SDS-PAGE analysis (W1, W2).

19. Elute HTNCre with 3 bed-volumes of elution buffer, take 10 ul of eluate fraction (E) for SDS-PAGE analysis.

20. Dialyse against High Salt Buffer twice (volume of dialysis buffer to sample is 50), first 1 hour, then O/N at 4 oC.

21. Dialyse against Gylcerol Buffer twice, first 3 ~ 4 hours, then O/N at 4 oC. This will result increase of Cre concentration to 4 folds

22. After dialysis HTCre concentration will be netween 200 and 450 μM, final yield will be ~12 mg per liter of expression culture.

24. Take 1 ul of glycerol stock (GS) for SDS-PAGE analysis.

25. Store HTNCre stock solution at -20°C or -80°C



{| border="1"
|-
! Material Name
! Type
! Company
! Catalogue Number
! Comment
|-
| TUNER (DE3) pLacI
| <br/>
| Novagen
| 70625
| <br/>
|-
| Glycerol
| <br/>
| Carl Roth
| 3783.2
| <br/>
|-
| Na<sub>2</sub>HPO<sub>4</sub>
| <br/>
| Roth
| T876.1
| <br/>
|-
| Trizma Base
| <br/>
| Sigma-Aldrich
| T1503
| <br/>
|-
| HCl
| <br/>
| Roth
| 4625.1
| <br/>
|-
| Imidazol
| <br/>
| Roth
| X998.4
| <br/>
|-
| NaCl
| <br/>
| Roth
| 9265.2
| <br/>
|-
| Yeast Extract
| <br/>
| Roth
| 2363.4
| <br/>
|-
| Trypton/Pepton
| <br/>
| Roth
| 8952.4
| <br/>
|-
| K<sub>2</sub>HPO<sub>4</sub>
| <br/>
| Roth
| P749.2
| <br/>
|-
| KH<sub>2</sub>PO<sub>4</sub>
| <br/>
| Roth
| 3904.1
| <br/>
|-
| Ampicillin
| <br/>
| Sigma
| A9518
| <br/>
|-
| Carbenicillin
| <br/>
| Sigma
| 6344.2
| <br/>
|-
| HEPES
| <br/>
| Sigma
| H3375
| <br/>
|-
| Lysozyme
| <br/>
| Sigma
| 62971
| <br/>
|-
| Benzonase
| <br/>
| Novagen
| <br/>
| <br/>
|-
| L-Tartaric acid, disodium salt
| <br/>
| Sigma
| <br/>
| <br/>
|-
| 50% Ni-NTA slurry
| <br/>
| Invitrogen
| R901-15
| <br/>
|-
| EconoPac columns
| <br/>
| Biorad
| 732-1010
| <br/>
|-
| Sterile filter 0,22μm
| <br/>
| Whatman
| <br/>
| <br/>
|-
| Paraformaldehyde (PFA)
| <br/>
| Sigma
| <br/>
| <br/>
|-
| LB medium
| <br/>
| <br/>
| <br/>
| Yeast extract, Trypton/Pepton, NaCl
|-
| TB medium
| <br/>
| <br/>
| <br/>
| 
Yeast extract, Trypton/Pepton, Glycerol, K<sub>2</sub>HPO<sub>4</sub>, KH<sub>2</sub>PO<sub>4</sub>

|-
| Lysis Buffer (PTB)
| <br/>
| <br/>
| <br/>
| 50 mM Na<sub>2</sub>HPO<sub>4</sub>, 5 mM Tris, pH 7.8
|-
| Tartaric Salt Buffer (TSB)
| <br/>
| <br/>
| <br/>
| PTB containing 2 M L-Tartaric acid, disodium salt, and 20 mM Imidazol
|-
| Washing Buffer
| <br/>
| <br/>
| <br/>
| PTB, 500 mM NaCl, 15 mM Imidazol
|-
| Elution Buffer
| <br/>
| <br/>
| <br/>
| PTB, 500 mM NaCl, 250 mM Imidazol
|-
| High Salt Buffer
| <br/>
| <br/>
| <br/>
| 600 mM NaCl, 20 mM HEPES, pH 7.4
|-
| Gylcerol Buffer
| <br/>
| <br/>
| <br/>
| 50% glycerol, 500 mM NaCl, 20 mM HEPES, pH 7.4
|-
| TrypLE™ Express
| <br/>
| Invitrogen
| <br/>
| <br/>
|-
| ESGRO (LIF)
| <br/>
| Millipore
| <br/>
| <br/>
|-
| NEAA
| <br/>
| Gibco
| 11140035
| <br/>
|-
| L-Glutamin
| <br/>
| Gibco
| 25030024
| <br/>
|-
| β-Mercaptoethanol
| <br/>
| Gibco
| 31350010
| <br/>
|-
| DMEM
| <br/>
| Gibco
| 11960044
| <br/>
|-
| PBS
| <br/>
| Gibco
| <br/>
| <br/>
|-
| Fetal Calf Serum (FCS)
| <br/>
| PAA
| <br/>
| <br/>
|-
| X-Gal staining solution:
| <br/>
| <br/>
| <br/>
| 4 mM K<sub>3</sub>(FeIII(CN)<sub>6</sub>), 4 mM K<sub>4</sub>(FeII(CN)<sub>6</sub>),<br/>2mM MgCl<sub>2</sub> 0.4 mg/mL X-Gal solved in PBS
|-
| K<sub>3</sub>(FeIII(CN)<sub>6</sub>)
| <br/>
| Sigma
| P-3367
| <br/>
|-
| K<sub>4</sub> (FeII(CN)<sub>6</sub>)
| <br/>
| Sigma
| P-9387
| <br/>
|-
| MgCl<sub>2</sub>
| <br/>
| Sigma
| M8266
| <br/>
|-
| X-Gal
| <br/>
| Sigma
| B4252
| <br/>
|}



<br/>'''TB medium 900 ml'''

Tryptone 12 g<br/>Yeast extract 24g<br/>Glycerol 4 ml<br/>Deionized water or MiliQ water to 900ml



Mix well and autoclave for 20 min at 15 psi on liquid cycle, Store at room temp

'''Potassium salt 1000 ml'''

0.17M KH2PO4 and 0.72 M K2HPO4

KH2PO4 23.1g<br/>K2HPO4 125.4g<br/>Deionized water 900 ml

After the salt has completely dissolved, adjust volume to 1000 ml with D.I. water Sterilize by autoclaving for 20 min at 15 psi on liquid cycle<br/>Store at room temp; do not add to TB broth until needed.



'''Complete TB medium''': add 100 ml Potassium salt to 900 ml TB medium.



'''Protein transduction into murine embryonic stem (ES) cells'''

1. Seed ES cells carrying a conditional β-Galactosidase reporter construct&nbsp; as single cells using TrypLE™ Express for dissociation of adherent cells.

2. After 4 to 6 hours the cells had re-attached and remove the medium. (80% confluency)

3. Incubate ES cells with HTNCre-containing medium for 16 hours.

Dilute an appropriate amount of HTNCre protein (corresponding to 10 μM) from the glycerol stock with ES medium and then sterile filtrated (0.22 μm).

4. After protein transduction change medium back to normal growth medium.<br/>5. After two days wash cells with PBS and fixed with 4% Paraformaldehyde (PFA) for 10 minutes.<br/>6. Wash 2 times with PBS

7. Stain fixed cells with X-Gal staining staining solution and incubate over night at 37°



Important: The product we are selling online is Cre from pTriEx-HTNC (His-Tat-NLS-Cre, clone #298) or pET28b-Cre (clone #375). Both of them produce His-Tat-NLS-Cre.

<br/>If protein expression level is low, use plasmid to transform Rosetta BL21 (DE3) pLysS completent cells and grow bacterial on plate containing Chl/Car (for pTriEX-HTNC)&nbsp;or Chl/Kan (for pET28b-Cre). Pick a few colonies and grow in 5 ml medium containing Chl/Car (for pTriEX-HTNC) or Chl/Kan for pET28b-Cre), make a glycerol stock, use the rest of cells for protein expression.