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--- purification [2016/05/18 14:38] – troffer
+++ purification [2023/11/01 20:18] (current) – external edit 127.0.0.1
@@ Line 1: / Line 1: @@
-**__Purification Carm1-130-487__**
+===== Purification =====
+[[purification:storage|Flash-freezing of protein samples for long-term storage]]
-Sur 1l baculo. Sonication dans 50ml 50mM TrisHCl pH8, 250mM NaCl, 5% glycerol, 5mM tcep, 0.01% NP40 + anti-protéases.
+[[purification:limited_proteolysis|Limited proteolysis]]
-Centrifugation 30mn 18000rpm.
+[[purification:TEV|In-house TEV production]]
-Incubation EB + 2ml billes GSH-sépharose o/n 4°C. Lavages billes 2x15ml tampon.
-Digest tev 4h 30°C dans 2ml. Récup surnageant v=2ml, lavage 1 v=2ml, lavage 2 v=1ml. Pool S+L et concentration jusqu’à v=1.5ml.
-Injection sur GF S200 16/60 et élution 1ml/mn 20mM TrisHCl pH7.5, 50mM NaCl, 1mM tcep.