https://bsi.inscog.eu/ 2026-06-01T23:06:15+00:00 https://bsi.inscog.eu/ https://bsi.inscog.eu/lib/exe/fetch.php?media=wiki:dokuwiki.svg text/html 2023-11-01T20:19:04+00:00 Anonymous (anonymous@undisclosed.example.com) https://bsi.inscog.eu/doku.php?id=purification:tev:b_protocol&rev=1698869944&do=diff Culture et purification TEV 1. Culture * Electrotransformation BL21(DE3) avec plasmide pTEV * Etalement sur boite LB agar + Kan 30 µg/ml * Quelques colonies prélevées pour ensemencer préculture de 100 ml de LB + kanamycine 30 µg/ml * Culture de 3 x 1 litre de LB Kan 30 µg/ml ensemencée avec la préculture pour avoir DO initiale de 0.1 text/html 2023-11-01T20:19:04+00:00 Anonymous (anonymous@undisclosed.example.com) https://bsi.inscog.eu/doku.php?id=purification:tev:c_protocol&rev=1698869944&do=diff Cabrita TEV Purification protocol Two point mutations were introduced to produce the double mutant L56V/S135G. The mutations were introduced using the QuikChange Site Directed Mutagenesis kit (Stratagene). * The proteins were expressed in BL21pRIPL text/html 2023-11-01T20:19:02+00:00 Anonymous (anonymous@undisclosed.example.com) https://bsi.inscog.eu/doku.php?id=purification:tev:faq&rev=1698869942&do=diff TEV Protease FAQ ---------- What protease inhibitors are known not to affect TEV? PMSF and AEBSF (1mM), TLCK (1mM), Bestatin (1mg/ml), pepstatin A (1mM), EDTA (1mM), and E-64 (3mg/ml), “complete” protease inhibitor cocktail (Roche). Zinc will inhibit the activity of the enzyme at concentrations of 5 mM or greater. Reagents that react with cysteine (e.g., iodoacetamide) are potent inhibitors of TEV protease. text/html 2023-11-01T20:19:04+00:00 Anonymous (anonymous@undisclosed.example.com) https://bsi.inscog.eu/doku.php?id=purification:tev:s_protocol&rev=1698869944&do=diff TEV preparation - Séraphin Lab j-1 * Transform BL21/LysS strain on KanR with optimized TEV plasmid (stock pBS2440). j * Inoculate 25 ml medium A with one colony at the end of the afternoon. * Grow at 37°C. j+1 * Inoculate 1 L medium A with the 25 ml preculture. text/html 2023-11-01T20:19:04+00:00 Anonymous (anonymous@undisclosed.example.com) https://bsi.inscog.eu/doku.php?id=purification:tev:w_protocol&rev=1698869944&do=diff Expression & Purification of His-TEV(S219V)-Arg S. Cherry, J. E. Tropea, D. S. Waugh * BL21-RIL cells containing pRK793 are grown at 37 °C in L-broth containing 100 μg/ml ampicillin and 30 μg/ml chloramphenicol. * When the cells reach mid log phase (OD600~0.5), IPTG is added to a final concentration of 1 mM and the temperature is reduced to 30 °C.