Purification of GST Fusion Proteins by GSTrap - Revision history

Davebrid: removed table of contents

2012-07-24T17:55:12Z

removed table of contents

← Older revision		Revision as of 17:55, 24 July 2012
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			__NOTOC__
	==Bacteria Production and Induction==		==Bacteria Production and Induction==
	*Express and induce protein in culture under appropriate conditions:		*Express and induce protein in culture under appropriate conditions:

Davebrid: /* FPLC Run */ Added details about HPLC run

2012-07-24T17:53:02Z

FPLC Run: Added details about HPLC run

← Older revision		Revision as of 17:53, 24 July 2012
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	==FPLC Run==		==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)		#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==		==Post-FPLC==

Davebrid: Added initial protocol, need to add details about HPLC Run

2012-07-24T17:39:21Z

Added initial protocol, need to add details about HPLC Run

New page

==Bacteria Production and Induction==
*Express and induce protein in culture under appropriate conditions:
#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.

==Lysis and Preparation==
#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.

==FPLC Run==
#Fill trey with ice, or put PBS and Glutathione in ice bucket.
#Start the pump, by going to
#Rinse the leads with water and place in PBS (A) and Glutathione (B)

==Post-FPLC==
#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
[[Category:Protein Purification]]