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His-Tat-NLS-FLP-Purification
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<br/><font color="#252525" face="sans-serif"><span style="font-size: 14px;">E coli</span></font> {| border="1" cellspacing="1" cellpadding="1" style="width:500px;" |- | ID | Name | PI, MW | Antibiotics | Expression<br/> | Link |- | 1853<br/> | pET28b-yFLP,<br/> | <br/> | Kan<br/> | N | [http://www.excellgen.net/eln/index.php/1860_1860_1853_1518_1519_1919_1654_1901_1859_1859_1873_1873 http://www.excellgen.net/eln/index.php/1860_1860_1853_1518_1519_1919_1654_1901_1859_1859_1873_1873]<br/> |- | 1860<br/> | pT7A3-yFLP<br/> | <br/> | Kan<br/> | N | [http://www.excellgen.net/eln/index.php/1500_1903_1519_1919_1868_1873_1859_1860_1870_1884_gfp http://www.excellgen.net/eln/index.php/1500_1903_1519_1919_1868_1873_1859_1860_1870_1884_gfp]<br/> |- | 1951<br/> | pT7A3-HC-yFLP<br/> | <br/> | Amp<br/> | ? | [http://www.excellgen.net/eln/index.php/1951_1952 http://www.excellgen.net/eln/index.php/1951_1952]<br/> |- | 1812<br/> | pET28b-HNFlp-Ecoli<br/> | <br/> | <br/> | ? | [http://www.excellgen.net/eln/index.php/1951_1952 http://www.excellgen.net/eln/index.php/1951_1952]<br/> |- | 1870<br/> | pET28b-HTNFlp-ecoli<br/> | <br/> | <br/> | N | [http://www.excellgen.net/eln/index.php/1500_1903_1519_1919_1868_1873_1859_1860_1870_1884_gfp http://www.excellgen.net/eln/index.php/1500_1903_1519_1919_1868_1873_1859_1860_1870_1884_gfp]<br/> |- | 1853<br/> | pET28b-yFLP<br/> | <br/> | <br/> | N | [http://www.excellgen.net/eln/index.php/1860_1860_1853_1518_1519_1919_1654_1901_1859_1859_1873_1873 http://www.excellgen.net/eln/index.php/1860_1860_1853_1518_1519_1919_1654_1901_1859_1859_1873_1873]<br/> |- | 2268<br/> | pHCMC04-iFLP<br/> | <br/> | <br/> | <br/> | <br/> |- | 2385<br/> | pL6H-TRX-Tat-NLS-iFlp-6H | <br/> | <br/> | N | [http://www.excellgen.net/eln/index.php/757_1139_1577_2005_2361_2384_2385_2388_2389_2390_2392 http://www.excellgen.net/eln/index.php/757_1139_1577_2005_2361_2384_2385_2388_2389_2390_2392]<br/> |- | 2184<br/> | pMAL-c5E-R9-HA-iFlp | <br/> | <br/> | ? | [http://www.excellgen.net/eln/index.php/2107_1836_2038_2040_2080_2178_2179_2184_2082_2086_2087_2088 http://www.excellgen.net/eln/index.php/2107_1836_2038_2040_2080_2178_2179_2184_2082_2086_2087_2088]<br/> |- | 2220<br/> | pMal-p-MBP-Tat-NLS-iFLP-6H | <br/> | <br/> | N | [http://www.excellgen.net/eln/index.php/1948_1950_1993_1994_1998_2019_2023_2037_2220 http://www.excellgen.net/eln/index.php/1948_1950_1993_1994_1998_2019_2023_2037_2220]<br/> |- | 1884<br/> | pT7A30HTNFlp-ecoli | <br/> | <br/> | N | [http://www.excellgen.net/eln/index.php/1500_1903_1519_1919_1868_1873_1859_1860_1870_1884_gfp http://www.excellgen.net/eln/index.php/1500_1903_1519_1919_1868_1873_1859_1860_1870_1884_gfp]<br/> |- | 1808<br/> | pT7A3-6His-NLS-Flpo | <br/> | <br/> | <br/> | not done yet |- | 2383<br/> | pT7A3-6XHis-SMT3-Tat-NLS-iFlp-6H | <br/> | <br/> | insoluble? | [http://www.excellgen.net/eln/index.php/2381_2383_2386_expression_screening_2375_purification http://www.excellgen.net/eln/index.php/2381_2383_2386_expression_screening_2375_purification]<br/> |- | 2378<br/> | pT7A3-Cre | <br/> | <br/> | <br/> | not done yet |- | 2379<br/> | pT7A3-Cre-(Tev)-Tat-NLS-iFLP-6H | 9.63 / 93.1<br/> | <br/> | insoluble | [http://www.excellgen.net/eln/index.php/2290_2294_2360_2361_2379_2382_2385_1995_2038_1708 http://www.excellgen.net/eln/index.php/2290_2294_2360_2361_2379_2382_2385_1995_2038_1708]<br/> |- | 2179<br/> | pT7A3HC-6H-Tat-sc-MS2-Flpo | <br/> | <br/> | insoluble | [http://www.excellgen.net/eln/index.php/2107_1836_2038_2040_2080_2178_2179_2184_2082_2086_2087_2088 http://www.excellgen.net/eln/index.php/2107_1836_2038_2040_2080_2178_2179_2184_2082_2086_2087_2088]<br/> |- | 1951<br/> | pT7A3-HC-yFLP | <br/> | <br/> | ? | [http://www.excellgen.net/eln/index.php/1951_1952 http://www.excellgen.net/eln/index.php/1951_1952]<br/> |- | 1813<br/> | pT7A3-HNFlp-Ecoli | 9.53 / 50.6<br/> | <br/> | ? | <br/> |- | 2180<br/> | pT7A3-R9-HA-iFlp | <br/> | <br/> | insoluble | [http://www.excellgen.net/eln/index.php/2180_2130_2174_cre_2108s_2108u http://www.excellgen.net/eln/index.php/2180_2130_2174_cre_2108s_2108u]<br/> |- | 2409<br/> | pT7A3-Tat-dCre-(Tev)-Tat-NLS-iFLP-6H | <br/> | <br/> | <br/> | not done yet |- | 1952<br/> | pT7A3-Tat-Flpo | <br/> | <br/> | ? | [http://www.excellgen.net/eln/index.php/1951_1952 http://www.excellgen.net/eln/index.php/1951_1952]<br/> |- | 2381<br/> | pT7A3-TRX-Tat-NLS-iFlp-6H | <br/> | <br/> | Y | [http://www.excellgen.net/eln/index.php/2381_2383_2386_expression_screening_2375_purification http://www.excellgen.net/eln/index.php/2381_2383_2386_expression_screening_2375_purification]<br/> |- | 1860<br/> | pT7A3-yFLP | <br/> | <br/> | N | [http://www.excellgen.net/eln/index.php/1500_1903_1519_1919_1868_1873_1859_1860_1870_1884_gfp http://www.excellgen.net/eln/index.php/1500_1903_1519_1919_1868_1873_1859_1860_1870_1884_gfp]<br/> |- | 2203 | <span style="color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px; white-space: nowrap;">pCold-MBP-Tat-NLS-iFLP-6H</span><br/> | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">9.03/96.9</span><br/> | <font color="#252525" face="sans-serif"><span style="font-size: 14px;"> </span></font> | Y, insoluble | <font color="#252525" face="sans-serif"><span style="font-size: 14px;"> </span></font>[http://www.excellgen.net/eln/index.php/2085_2101_2146_2163_2187+2188_2190_2195_2203_20140512 http://www.excellgen.net/eln/index.php/2085_2101_2146_2163_2187%2B2188_2190_2195_2203_20140512] |- | 2115 | pT7A3-iFLP<br/> | <br/> | <br/> | Y, insloluble | [http://www.excellgen.net/eln/index.php/2115_2203_with_chaperones http://www.excellgen.net/eln/index.php/2115_2203_with_chaperones]<br/> |- | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2108</span><br/> | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">pCold-Tat-NLS-iFLP-6H</span><br/> | <br/> | <br/> | insloluble<br/> | [http://www.excellgen.net/eln/index.php/1708_1823_2116_2133_2115_2108 [1]]<br/> |} 2/17/19 [[File:2115 2108 1708 expression.jpg|RTENOTITLE]] Mammalian <div> </div> {| border="1" cellspacing="1" cellpadding="1" style="width:500px;" |- | ID | Name | <br/> | <br/> | <br/> |- | 471<br/> | pAAV.CMV-FLPe<br/> | <br/> | <br/> | <br/> |- | 494<br/> | pAd52-mCMV-MCS<br/> | <br/> | <br/> | <br/> |- | 1525<br/> | pCDNA3.1-FLPe | <br/> | <br/> | <br/> |- | 1526<br/> | pCDNA3.1-NLS-FLPo | <br/> | <br/> | <br/> |- | 2173<br/> | pCMV5-CD16-Tat-Flpo-RIgE | <br/> | <br/> | <br/> |- | 2173<br/> | pCMV5-CD16-Tat-Flpo-RIgE | <br/> | <br/> | <br/> |- | 2137<br/> | pCMV5-CD63-Flpe | <br/> | <br/> | <br/> |- | 2137<br/> | pCMV5-CD63-Flpe | <br/> | <br/> | <br/> |- | 2131<br/> | pCMV5-Gag-Flpe-IRES-VSVG-WPRE-oriP | <br/> | <br/> | <br/> |- | 2131<br/> | pCMV5-Gag-Flpe-IRES-VSVG-WPRE-oriP | <br/> | <br/> | <br/> |- | 1445<br/> | pCMV5-Gag-NLS-Flpe-WPRE-oriP | <br/> | <br/> | <br/> |- | 1969<br/> | pCMV5-Gag-p2b-NLS-Flpe-WPRE-oriP | <br/> | <br/> | <br/> |- | 1962<br/> | pCMV5-HTN-Flpo | <br/> | <br/> | <br/> |- | 2122<br/> | pCMV5-Mfge8-Tat-FLPo-MFGE8 | <br/> | <br/> | <br/> |- | 1964<br/> | pCMV5-n-NA-NLS-Flpe-WPRE-oriP | <br/> | <br/> | <br/> |- | 2139<br/> | pCMV5-pene-CD63-Flpe | <br/> | <br/> | <br/> |- | 2139<br/> | pCMV5-pene-CD63-Flpe | <br/> | <br/> | <br/> |- | 2140<br/> | pCMV5-tat-CD63-Flpe | <br/> | <br/> | <br/> |- | 2140<br/> | pCMV5-tat-CD63-Flpe | <br/> | <br/> | <br/> |- | 1981<br/> | pCMV5-Tat-Flpo-PDGFR | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |} Baculovirus <div> </div> {| border="1" cellspacing="1" cellpadding="1" style="width:500px;" |- | ID | Name | <br/> | <br/> | <br/> |- | 471<br/> | pAAV.CMV-FLPe<br/> | <br/> | <br/> | <br/> |- | 494<br/> | pAd52-mCMV-MCS<br/> | <br/> | <br/> | <br/> |- | 2219<br/> | pFastbacDual-p10-VSVG-M-6H-Tat-Flp-MFGE8 | <br/> | <br/> | <br/> |- | 2320<br/> | pFastBacDual-VSVG-Flpo | <br/> | <br/> | <br/> |- | 2227<br/> | pFastBac-Dual-VSVG-HIVGag-FLP-CD63-Flpe | <br/> | <br/> | <br/> |- | 2226<br/> | pFastBac-Dual-VSVG-HIVGag-FLPe | <br/> | <br/> | <br/> |- | 2225<br/> | pFastbacDual-VSVG-M-6H-Tat-Flp-MFGE8-Gag-Flpe | <br/> | <br/> | <br/> |- | 2352<br/> | pFastbac-GP64-Cre-Flpe-WPRE | <br/> | <br/> | <br/> |- | 2354<br/> | pFastbac-GP64-Flpe-WPRE | <br/> | <br/> | <br/> |- | 2353<br/> | pFastbac-GP64-Flp-Flpe-WPRE | <br/> | <br/> | <br/> |- | 2364<br/> | pFastBac-oriP-Flpo-PDGFR-WPRE | <br/> | <br/> | <br/> |- | 2410<br/> | pFastBac-VSVG-CMV5-Flp | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |} mRNA {| border="1" cellspacing="1" cellpadding="1" style="width:500px;" |- | ID | Name | <br/> | <br/> | <br/> |- | 963 | pMiniT2-Flpo<br/> | <br/> | <br/> | <br/> |- | 960 | pT7CFE1-NHis-GST-His-Tat-NLS-Flpo-CH<br/> | <br/> | <br/> | <br/> |- | 2150<br/> | pEC-Flpo-MS2Pac | <br/> | <br/> | <br/> |- | 2150<br/> | pEC-Flpo-MS2Pac | <br/> | <br/> | <br/> |- | 2151<br/> | pEC-Flpo-PP7Pac | <br/> | <br/> | <br/> |- | 2151<br/> | pEC-Flpo-PP7Pac | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |} VLP {| border="1" cellspacing="1" cellpadding="1" style="width:500px;" |- | ID | Name | <br/> | <br/> | <br/> |- | 471<br/> | pAAV.CMV-FLPe<br/> | <br/> | <br/> | <br/> |- | 494<br/> | pAd52-mCMV-MCS<br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |} <div>Yeast</div><div> {| border="1" cellspacing="1" cellpadding="1" style="width:500px;" |- | ID | Name | <br/> | <br/> | <br/> |- | 1854<br/> | pPinkalpha-HC-yFLP | <br/> | <br/> | <br/> |- | 2159<br/> | pJ912-MS2Pac-Flpo | <br/> | <br/> | <br/> |- | 2159<br/> | pJ912-MS2Pac-Flpo | <br/> | <br/> | <br/> |- | 1976<br/> | pJ912-yFLP | <br/> | <br/> | <br/> |- | 2120<br/> | pJP1520-CD63 | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <br/> | <br/> | <br/> | <br/> |} <br/></div><div><br/></div><div><br/></div><div></div><div><br/></div> 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 [[File:FLP exp BL21 strains 1 2014.jpg|RTENOTITLE]]<br/>1853 pET28b-yFLP, Kan<br/>1860 pT7A3-yFLP, Kan<br/>1951 pT7A3-HC-yFLP, Amp<br/>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.<br/>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.<br/>5. Grow to OD600 to 1.5 at 28 oC.<br/>6. Add IPTG at a final concentration of 0.5 mM and to induce for 1 hour at 28 oC.<br/>7. Spin down cells and store pellet at –20 oC.<br/>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.<br/>9. Lyse cells by adding 2 mg/ml lysozyme, 25 U/ml benzonase<br/>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.<br/>11. Spin for 30 minutes at 25000 rpm at 4 oC.<br/>12. Load cleared lysate to Ni-NTA agarose column (1.5 ml of slurry per 1 liter of bacterial expression culture.<br/>13. Wash beads with '''Washing Buffer I''', 50 mM NaH2PO4, 1 M NaCl, 20 mM imidazole until protein is not detectable at OD280<br/>14. Wash beads with 3 CV of '''Washing Buffer II''', 50 mM NaH2PO4, 1 M NaCl, 5% glycerol, 20 mM imidazole.<br/>14. Elute with 5 CV of '''Elution Buffer''', 50 mM NaH2PO4, pH 7.4, 300 mM NaCl, 250 mM imidazole, 5% glycerol.<br/>15. Dilute eluted protein with 3 volume of '''SP Buffer''', 50 mM NaH2PO4, pH 7.4, 5% glycerol.<br/>16. Load to 5 ml Pharmacia Q FF cartridge + 15 ml SP FF column (Pharmacia column is in front of SP FF column).<br/>17. Disconnect (remove) Q FF, Wash SP FF column with '''SP Buffer '''50 mM NaH2PO4, pH 7.4, until OD280 reaches baseline.<br/>18. Elute with linear gradient of 0.1 to 1 M NaCl in '''SP Buffer '''<br/>19. Combine fractions containing FLP. Concentrate protein using spin column if concentration is low.<br/>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).<br/>21. Filter through a 0.22 μm filter.<br/>22. Measure protein concentration, aliquote 1 mg to autoclaved vials. Label tubes and store at -80 oC.<br/>23. Input data into protein database.<br/>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 <br/>Use these links to calculate amount of protein required for transduction [http://www.molbiol.ru/eng/scripts/01_04.html http://www.molbiol.ru/eng/scripts/01_04.html] [http://www.my-whiteboard.com/pub/html/mw_2_moles.html http://www.my-whiteboard.com/pub/html/mw_2_moles.html] [[Category:Protocols]] [[Category:Products]]
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