Difference between revisions of "Purification of GST Fusion Proteins"

Latest revision as of 14:40, 12 August 2009

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 overnight culture to 1L TB/Amp and grow at 37C
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.

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 (i.e. 20,000RMP in JA25.5 at 4 degrees C) for 30 min to clarify
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 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) or other desired buffer
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 purified protein (0.5-10 ug) by SDS-PAGE

@@ Line 16: / Line 16: @@
 #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 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
-*Elution buffer contains 50mM Glutathionie in PBS(0.615g in 40mL PBS), pH to between 7 and 8.
+#Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X) or other desired buffer
-#Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X)
+#Measure protein concentration ([[Bradford Assay]] or [[Quantification by Absorbance at 280nm]]; concentrate if necessary and store at -20)
-#Measure protein concentration (Bradford or A280; 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 purified 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 protein (0.5-10 ug) by SDS-PAGE
 [[Category:Protein Purification]]