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#*Express and induce protein in culture under appropriate conditions (normally do the following):#grow an overnight culture in ~25 mL LB/Amp (+with Chloramphenicol if neededusing 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 10 100 uM IPTG (optimize the concentration of IPTG and duration of induction should be optimized for each protein)
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==Bacteria Production and Induction==
#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 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 (i.e. 20,000RMP in JA25.5 at 4 degrees C) for 30 min to clarify#Add ~01.5 0 mL glutathione-agarose to 50 mL PBS (no PI) to prepare beads. Pellet 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 5 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 A280[[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]]