Difference between revisions of "Purification of GST Fusion Proteins by GSTrap"

Revision as of 17:53, 24 July 2012

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
Prepare, filter and chill 1L of PBS and ~50 mL of 10 mM Glutathione (pH 8.0) in PBS.

Fill tray with ice, or put PBS and Glutathione in ice bucket.
Turn computer on and login if necessary, username=saltielhplc; password=eatAdEv3
Open TC Navigator and login
Go to Apps -> Kickoff -> GST Purification
Enter number of samples you want to run
Enter sample names and click through to finish
Start the pump, by going to Hands On, Start Pump
Turn on lamp if necessary by going to Detector -> Turn Lamp On within the Hands On Window
Rinse the leads with water and place in PBS (A) and Glutathione (B)
Prime the leads by first selecting 100% A on the Hands On Window and while the pump is running, loosen the black screw in the lower section (remove the panel). Attach a 50 mL syringe and pull through 5-10mL
Switch the pump to 100% B (Glutathione solution) and prime those lines. Switch back to 100% A (PBS)
Connect the GSTrap column and let equillibrate for a few minutes
WIth the injection loop set to load, using a syringe inject ~5mL of lysate into the 5mL sample loop
Turn the loop clockwise to inject to start the run
After 30min, the fraction collecter will automatically start, make sure it has 2mL tubes without caps ready

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 14: / Line 14: @@
 ==FPLC Run==
-#Fill trey with ice, or put PBS and Glutathione in ice bucket.
+#Fill tray with ice, or put PBS and Glutathione in ice bucket.
-#Start the pump, by going to
+#Turn computer on and login if necessary, username=saltielhplc; password=eatAdEv3
+#Open TC Navigator and login
+#Go to Apps -> Kickoff -> GST Purification
+#Enter number of samples you want to run
+#Enter sample names and click through to finish
+#Start the pump, by going to Hands On, Start Pump
+#Turn on lamp if necessary by going to Detector -> Turn Lamp On within the Hands On Window
 #Rinse the leads with water and place in PBS (A) and Glutathione (B)
+#Prime the leads by first selecting 100% A on the Hands On Window and while the pump is running, loosen the black screw in the lower section (remove the panel).  Attach a 50 mL syringe and pull through 5-10mL
+#Switch the pump to 100% B (Glutathione solution) and prime those lines.  Switch back to 100% A (PBS)
+#Connect the GSTrap column and let equillibrate for a few minutes
+#WIth the injection loop set to load, using a syringe inject ~5mL of lysate into the 5mL sample loop
+#Turn the loop clockwise to inject to start the run
+#After 30min, the fraction collecter will automatically start, make sure it has 2mL tubes without caps ready
 ==Post-FPLC==