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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 ...'
==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
#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