Purification of GST Fusion Proteins - Revision history

Davebrid at 14:40, 12 August 2009

2009-08-12T14:40:13Z

← Older revision		Revision as of 14:40, 12 August 2009
Line 19:		Line 19:
	#Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions. Check fractions for protein with Bradford assay		#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		#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 [[Quantification by Absorbance at 280nm]]; 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 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 purified protein (0.5-10 ug) by SDS-PAGE
	[[Category:Protein Purification]]		[[Category:Protein Purification]]

Davebrid at 14:39, 12 August 2009

2009-08-12T14:39:50Z

← Older revision		Revision as of 14:39, 12 August 2009
Line 19:		Line 19:
	#Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions. Check fractions for protein with Bradford assay		#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		#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 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]]		[[Category:Protein Purification]]

Davebrid at 14:39, 12 August 2009

2009-08-12T14:39:05Z

← Older revision		Revision as of 14:39, 12 August 2009
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		#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		#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		#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 or A280; 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 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]]		[[Category:Protein Purification]]

Davebrid at 14:37, 12 August 2009

2009-08-12T14:37:34Z

← Older revision		Revision as of 14:37, 12 August 2009
Line 21:		Line 21:
	#Measure protein concentration (Bradford or A280; 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 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]]

Davebrid: /* Bacteria Production and Induction */

2009-08-12T14:34:56Z

Bacteria Production and Induction

← Older revision		Revision as of 14:34, 12 August 2009
Line 1:		Line 1:
	==Bacteria Production and Induction==		==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]]).		#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==		==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		#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

Jpecherer: Updates to previous version, updatite the lysis and purification section.

2009-05-07T15:39:36Z

Updates to previous version, updatite the lysis and purification section.

Davebrid at 15:12, 6 May 2009

2009-05-06T15:12:24Z

← Older revision		Revision as of 15:12, 6 May 2009
Line 4:		Line 4:
	#add 5 mL culture to 1L TB/Amp and grow at 37C		#add 5 mL 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 10 uM IPTG (optimize for each protein)
	#let grow O/N at <25C (optimize induction time/temp 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==		==Lysis and Purification==

Davebrid: Created page with '==Bacteria Production and Induction== #Express and induce protein in culture under appropriate conditions (normally do the following) #grow an overnight culture in ~25 mL LB/Amp ...'

2009-05-05T15:07:57Z

Created page with '==Bacteria Production and Induction== #Express and induce protein in culture under appropriate conditions (normally do the following) #grow an overnight culture in ~25 mL LB/Amp ...'

New page

==Bacteria Production and Induction==
#Express and induce protein in culture under appropriate conditions (normally do the following)
#grow an overnight culture in ~25 mL LB/Amp (+Chloramphenicol if needed) from a colony <2 weeks post transformation
#add 5 mL 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)
#let grow O/N at <25C (optimize induction time/temp for each protein).
#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
#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
#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
#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)
#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 protein (0.5-10 ug) by SDS-PAGE

← Older revision		Revision as of 15:39, 7 May 2009
Line 1:		Line 1:
	==Bacteria Production and Induction==		==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 (normally innoculate the previous day)
	#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 (+Chloramphenicol if needed) from a colony <2 weeks post transformation
	#add 5 mL culture to 1L TB/Amp and grow at 37C		#add 5 mL culture to 1L TB/Amp and grow at 37C
Line 9:		Line 9:
	#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		#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)		#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		#Save a lysate sample (20 uL) and add clarified lysate to equilibrated beads
	#Incubate with rotation for 1h at 4C		#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		#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
	#Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions. Check fractions for protein with Bradford assay		#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)		#Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X)
	#Measure protein concentration (Bradford or A280; 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 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