Purification of GST Fusion Proteins: Difference between revisions

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==Bacteria Production and Induction==

#Express and induce protein in culture under appropriate conditions ~~(normally innoculate the previous day)~~

*Express and induce protein in culture under appropriate conditions:

#grow an overnight culture in ~25 mL LB/Amp (+Chloramphenicol if ~~needed~~) from a colony <2 weeks post transformation

#grow an overnight culture in ~25 mL LB/Amp (with Chloramphenicol if using Rosetta cells) from a colony <2 weeks post transformation.

#add 5 mL culture to 1L TB/Amp and grow at 37C

#add 5 mL overnight culture to 1L TB/Amp and grow at 37C

#grow to OD600 of 0.6-1.0 and induce with 10 uM IPTG (~~optimize~~ for each protein)

#grow to OD600 of 0.6-1.0 and induce with 100 uM IPTG (the concentration of IPTG and duration of induction should be optimized for each protein)

#let grow O/N at <25C (optimize induction time/temp for each protein, see [[Induction Conditions]]).

#centrifuge cells 10 min at 9000 RPM to pellet bacteria freeze if stopping at this point

#centrifuge cells 10 min at 9000 RPM to pellet bacteria freeze if stopping at this point.

==Lysis and Purification==

#Resuspend cells in ~20 mL lysis buffer (PBS + 0.1% B-ME and a PI tablet). Freeze in liquid nitrogen if not continuing with purification

@@ Line 1: / Line 1: @@
 ==Bacteria Production and Induction==
-#Express and induce protein in culture under appropriate conditions (normally innoculate the previous day)
+*Express and induce protein in culture under appropriate conditions:
-#grow an overnight culture in ~25 mL LB/Amp (+Chloramphenicol if needed) from a colony <2 weeks post transformation
+#grow an overnight culture in ~25 mL LB/Amp (with Chloramphenicol if using Rosetta cells) from a colony <2 weeks post transformation.
-#add 5 mL culture to 1L TB/Amp and grow at 37C
+#add 5 mL overnight culture to 1L TB/Amp and grow at 37C
-#grow to OD600 of 0.6-1.0 and induce with 10 uM IPTG (optimize for each protein)
+#grow to OD600 of 0.6-1.0 and induce with 100 uM IPTG (the concentration of IPTG and duration of induction should be optimized for each protein)
 #let grow O/N at <25C (optimize induction time/temp for each protein, see [[Induction Conditions]]).
-#centrifuge cells 10 min at 9000 RPM to pellet bacteria freeze if stopping at this point
+#centrifuge cells 10 min at 9000 RPM to pellet bacteria freeze if stopping at this point.
 ==Lysis and Purification==
 #Resuspend cells in ~20 mL lysis buffer (PBS + 0.1% B-ME and a PI tablet).  Freeze in liquid nitrogen if not continuing with purification