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--- purification:tev:s_protocol [2016/07/13 15:33] – created bonnefon
+++ purification:tev:s_protocol [2023/11/01 20:19] (current) – external edit 127.0.0.1
@@ Line 2: / Line 2: @@
 **j-1**
-Transform BL21/LysS strain on KanR with optimized TEV plasmid (stockpBS2440).
+  * 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.
+  * Inoculate 25 ml medium A((LB, Cm 25 μg/ml, Kan 10 μg/ml)) with one colony at the end of the afternoon.
-Grow at 37°C.
+  * Grow at 37°C.
 **j+1**
-Inoculate 1 L medium A with the 25 ml preculture.
+  * Inoculate 1 L medium A with the 25 ml preculture.
-Grow at 30°C until OD600 reach 0.9.
+  * Grow at 30°C until OD600 reach 0.9.
-Add IPTG to the medium up to 0.1 mM.
+  * Add IPTG to the medium up to 0.1 mM.
-Grow 6 hours at 23 °C.
+  * Grow 6 hours at 23 °C.
-Pellet cells 20 min at 4,000 rpm  in GS3.
+  * Pellet cells 20 min at 4,000 rpm  in GS3.
-Wash with PBS 1X.
+  * Wash with PBS 1X.
-Freeze pellet in liquid N2 and store at -80°C.
+  * Freeze pellet in liquid N2 and store at -80°C.
 **j+2**
-Resuspend pellet in Buffer 1.
+  * Resuspend pellet in Buffer 1((20 mM Tris-HCl pH 8.0, 10 mM imidazole, 200 mM Nacl, 2 mM β-ME, 0.2% NP40))
-Adjust to 20 ml with buffer 1.
+  * Adjust to 20 ml with Buffer 1.
-Pass through French-Press 2 times.
+  * Pass through French-Press 2 times.
-Spin down with ss34 12,000 rpm for 30 min.
+  * Spin down with ss34 12,000 rpm for 30 min.
-Put the supernatant with 1 ml slurry of NiNTA beads (previously washed with buffer 1) in a Falcon tube.
+  * Put the supernatant with 1 ml slurry of NiNTA beads (previously washed with buffer 1) in a Falcon tube.
-Rotate 1h at 4°C.
+  * Rotate 1h at 4°C.
-Transfer in an Econo-column (Biorad).
+  * Transfer in an Econo-column (Biorad).
-Wash with 15ml buffer 1.
+  * Wash with 15ml Buffer 1.
-Wash with 5 ml buffer 2.
+  * Wash with 5 ml Buffer 2((20 mM Tris-HCl pH 8.0, 10 mM imidazole, 1 M NaCl, 2 mM β-ME, 0.2% NP40))
-wash with 10 ml buffer 1.
+  * wash with 10 ml Buffer 1.
-Elute with 4x0.5 ml buffer 3 in 0.5 ml glycerol tube (50% glycerol final).
+  * Elute with 4x0.5 ml Buffer 3((20 mM Tris-HCl pH 8.0, 200 mM imidazole, 200 mM NaCl, 2 mM β-ME, 0.2% NP40)) in 0.5 ml glycerol tube (50% glycerol final).
-store at -80°C.
+  * store at -80°C.
 You may perform an additional chromatography step fort higher purity.