His-Tat-NLS-FLP-Purification
E coli
2/17/19
Mammalian
| ID | Name | |||
| 471 |
pAAV.CMV-FLPe |
|||
| 494 |
pAd52-mCMV-MCS |
|||
| 1525 |
pCDNA3.1-FLPe | |||
| 1526 |
pCDNA3.1-NLS-FLPo | |||
| 2173 |
pCMV5-CD16-Tat-Flpo-RIgE | |||
| 2173 |
pCMV5-CD16-Tat-Flpo-RIgE | |||
| 2137 |
pCMV5-CD63-Flpe | |||
| 2137 |
pCMV5-CD63-Flpe | |||
| 2131 |
pCMV5-Gag-Flpe-IRES-VSVG-WPRE-oriP | |||
| 2131 |
pCMV5-Gag-Flpe-IRES-VSVG-WPRE-oriP | |||
| 1445 |
pCMV5-Gag-NLS-Flpe-WPRE-oriP | |||
| 1969 |
pCMV5-Gag-p2b-NLS-Flpe-WPRE-oriP | |||
| 1962 |
pCMV5-HTN-Flpo | |||
| 2122 |
pCMV5-Mfge8-Tat-FLPo-MFGE8 | |||
| 1964 |
pCMV5-n-NA-NLS-Flpe-WPRE-oriP | |||
| 2139 |
pCMV5-pene-CD63-Flpe | |||
| 2139 |
pCMV5-pene-CD63-Flpe | |||
| 2140 |
pCMV5-tat-CD63-Flpe | |||
| 2140 |
pCMV5-tat-CD63-Flpe | |||
| 1981 |
pCMV5-Tat-Flpo-PDGFR | |||
Baculovirus
| ID | Name | |||
| 471 |
pAAV.CMV-FLPe |
|||
| 494 |
pAd52-mCMV-MCS |
|||
| 2219 |
pFastbacDual-p10-VSVG-M-6H-Tat-Flp-MFGE8 | |||
| 2320 |
pFastBacDual-VSVG-Flpo | |||
| 2227 |
pFastBac-Dual-VSVG-HIVGag-FLP-CD63-Flpe | |||
| 2226 |
pFastBac-Dual-VSVG-HIVGag-FLPe | |||
| 2225 |
pFastbacDual-VSVG-M-6H-Tat-Flp-MFGE8-Gag-Flpe | |||
| 2352 |
pFastbac-GP64-Cre-Flpe-WPRE | |||
| 2354 |
pFastbac-GP64-Flpe-WPRE | |||
| 2353 |
pFastbac-GP64-Flp-Flpe-WPRE | |||
| 2364 |
pFastBac-oriP-Flpo-PDGFR-WPRE | |||
| 2410 |
pFastBac-VSVG-CMV5-Flp | |||
mRNA
| ID | Name | |||
| 963 | pMiniT2-Flpo |
|||
| 960 | pT7CFE1-NHis-GST-His-Tat-NLS-Flpo-CH |
|||
| 2150 |
pEC-Flpo-MS2Pac | |||
| 2150 |
pEC-Flpo-MS2Pac | |||
| 2151 |
pEC-Flpo-PP7Pac | |||
| 2151 |
pEC-Flpo-PP7Pac | |||
VLP
| ID | Name | |||
| 471 |
pAAV.CMV-FLPe |
|||
| 494 |
pAd52-mCMV-MCS |
|||
| ID | Name | |||
| 1854 |
pPinkalpha-HC-yFLP | |||
| 2159 |
pJ912-MS2Pac-Flpo | |||
| 2159 |
pJ912-MS2Pac-Flpo | |||
| 1976 |
pJ912-yFLP | |||
| 2120 |
pJP1520-CD63 | |||
1. Transform Escherichia coli BL21 (DE3)/pRare2pLysS with yeast FLP plasmid
BL21 cells: T7 Express, BL21star(DE3), Nico21(DE3), Rosetta(DE3)
1/24/2014
Competent cells & vectors
1853 pET28b-yFLP, Kan
1860 pT7A3-yFLP, Kan
1951 pT7A3-HC-yFLP, Amp
2. Grow cells in LB containing 25 ug/ml carbenicillin or 50 ug/ml Kan, Chl and 0.2 % glucose o/v at 37 oc.
3. Inoculate 1000 ml cultures of TB medium containing 0.2% glucose, Chl and 100 ug/ml ampicillin or 50 ug/ml Kan with overnight cultures at 1:25 ratio
TB
Tryptone, 12 g
Yeast extract, 24 g
Glycerol, 4 mL
2 g glucose
Add Deionized H2O to 900 mL
Shake until the solutes have dissolved and then sterilize by autoclave
Allow the solution to cool to 60°C or less, and then add 100 mL of a sterile solution of 0.17 M KH2PO4, 0.72 M K2HPO4.
0.17 M KH2PO4, 0.72 M K2HPO4
Dissolve 23.1 g of KH2PO4 and 125.4 g of K2HPO4 in 900 mL of H2O. After the salts have dissolved, adjust the volume of the solution to 1000 mL with H2O, autoclave.
4. Grow at 37 oC until an OD600 of 1.0.
5. Grow to OD600 to 1.5 at 28 oC.
6. Add IPTG at a final concentration of 0.5 mM and to induce for 1 hour at 28 oC.
7. Spin down cells and store pellet at –20 oC.
8. Thaw cell pellets in 10 ml Lysis Buffer, 500 mM NaH2PO4, 50 mM Tris, pH 7.8 per 1 liter of bacterial expression culture.
9. Lyse cells by adding 2 mg/ml lysozyme, 25 U/ml benzonase
10. Add an equal volume of Salt Buffer, 2 M NaCl, 50 mM NaH2PO4, 50 mM Tris, pH 7.8, 20 mM imidazole, then sonicate.
11. Spin for 30 minutes at 25000 rpm at 4 oC.
12. Load cleared lysate to Ni-NTA agarose column (1.5 ml of slurry per 1 liter of bacterial expression culture.
13. Wash beads with Washing Buffer I, 50 mM NaH2PO4, 1 M NaCl, 20 mM imidazole until protein is not detectable at OD280
14. Wash beads with 3 CV of Washing Buffer II, 50 mM NaH2PO4, 1 M NaCl, 5% glycerol, 20 mM imidazole.
14. Elute with 5 CV of Elution Buffer, 50 mM NaH2PO4, pH 7.4, 300 mM NaCl, 250 mM imidazole, 5% glycerol.
15. Dilute eluted protein with 3 volume of SP Buffer, 50 mM NaH2PO4, pH 7.4, 5% glycerol.
16. Load to 5 ml Pharmacia Q FF cartridge + 15 ml SP FF column (Pharmacia column is in front of SP FF column).
17. Disconnect (remove) Q FF, Wash SP FF column with SP Buffer 50 mM NaH2PO4, pH 7.4, until OD280 reaches baseline.
18. Elute with linear gradient of 0.1 to 1 M NaCl in SP Buffer
19. Combine fractions containing FLP. Concentrate protein using spin column if concentration is low.
20. Dialyse against Dialysis Buffer (2 M NaCl, 50 mM HEPES or Na-Phosphate pH 7.4, 1 mM DTT, 1 mM EDTA, and 50% glycerol).
21. Filter through a 0.22 μm filter.
22. Measure protein concentration, aliquote 1 mg to autoclaved vials. Label tubes and store at -80 oC.
23. Input data into protein database.
24. FLP activity assay using FRT cell line or 293E cells transfected with FRT reporter
1 uM is about 50 μg Tat-Flp in ml medium. 4 μM is 200 ug in 1 ml medium. 4 μM should induce >60% recombination
HTN-FLP pI/Mw: 9.76 / 52.8
Use these links to calculate amount of protein required for transduction