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 do the following)~~

*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

#French Press cells 2 x 15 000 psi (see French Press Protocol)

#Centrifuge lysate at >15 000 RPM for 30 min to clarify

#Centrifuge lysate at >15 000 RPM (i.e. 20,000RMP in JA25.5 at 4 degrees C) for 30 min to clarify

#Add ~0.5 mL glutathione-agarose to 50 mL PBS (no PI) to prepare beads. ~~Pellet~~ beads 5 min at 1000 RPM and remove buffer

#Add 1.0 mL glutathione-agarose to 50 mL PBS (no PI) to prepare beads. Let sit for 30 min. to allow beads to settle or pellet beads for 5 min at 1000 RPM and remove buffer

#Save a lysate sample (20 uL) and add clarified lysate to equilibrated beads

#Incubate with rotation for 1h at 4C

#Pellet beads (save sample of unbound protein) and wash 3 x 50 mL cold wash buffer being careful to not suck up beads

#Pour into disposable column (BioRad # 732-6008) to collect beads. Wash with another 5 mL of PBS

#Pour into disposable column (BioRad # 732-6008) to collect beads. Wash with another 10 mL of PBS

#Prepare elution buffer containing 50mM Glutathionie in PBS(0.615g in 40mL PBS), adjust pH to between 7 and 8.

#Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions. Check fractions for protein with Bradford assay

#Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X)

#Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X) or other desired buffer

#Measure protein concentration (Bradford or ~~A280~~; concentrate if necessary and store at -20)

#Measure protein concentration ([[Bradford Assay]] or [[Quantification by Absorbance at 280nm]]; concentrate if necessary and store at -20)

#Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and protein (0.5-10 ug) by SDS-PAGE

#Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and purified protein (0.5-10 ug) by SDS-PAGE

[[Category:Protein Purification]]

@@ Line 1: / Line 1: @@
 ==Bacteria Production and Induction==
-#Express and induce protein in culture under appropriate conditions (normally do the following)
+*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
 #French Press cells 2 x 15 000 psi (see French Press Protocol)
-#Centrifuge lysate at >15 000 RPM for 30 min to clarify
+#Centrifuge lysate at >15 000 RPM (i.e. 20,000RMP in JA25.5 at 4 degrees C) for 30 min to clarify
-#Add ~0.5 mL glutathione-agarose to 50 mL PBS (no PI) to prepare beads.  Pellet beads 5 min at 1000 RPM and remove buffer
+#Add 1.0 mL glutathione-agarose to 50 mL PBS (no PI) to prepare beads. Let sit for 30 min. to allow beads to settle or pellet beads for 5 min at 1000 RPM and remove buffer
 #Save a lysate sample (20 uL) and add clarified lysate to equilibrated beads
 #Incubate with rotation for 1h at 4C
 #Pellet beads (save sample of unbound protein) and wash 3 x 50 mL cold wash buffer being careful to not suck up beads
-#Pour into disposable column (BioRad # 732-6008) to collect beads.  Wash with another 5 mL of PBS
+#Pour into disposable column (BioRad # 732-6008) to collect beads.  Wash with another 10 mL of PBS
+#Prepare elution buffer containing 50mM Glutathionie in PBS(0.615g in 40mL PBS), adjust pH to between 7 and 8.
 #Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions.  Check fractions for protein with Bradford assay
-#Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X)
+#Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X) or other desired buffer
-#Measure protein concentration (Bradford or A280; concentrate if necessary and store at -20)
+#Measure protein concentration ([[Bradford Assay]] or [[Quantification by Absorbance at 280nm]]; concentrate if necessary and store at -20)
-#Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and protein (0.5-10 ug) by SDS-PAGE
+#Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and purified protein (0.5-10 ug) by SDS-PAGE
+[[Category:Protein Purification]]