915
edits
Changes
Added initial protocol, need to add details about HPLC Run
==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]]
*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]]
