Grow cells at 30°C.
Innoculate 1L of LB or TB + Amp with 10mL of overnight culture.
Induce cells with 0.5mM IPTG at OD ~0.7
After ~ 3 hrs harvest cells by centrifugation, 20m x 4krpm
Freeze cells (-80°C) with 5mL H6 protein lysis buffer.
Thaw with additional 5mL lysis buffer and protease inhibitor (Roche EDTA free protease inhibitor capsules work well)
Lyse by your favorite method
Centrifuge- 30m x 15krpm
Ni-NTA column pre-incubate supernatant with washed Ni2+-NTA slurry. (~30m with shaking in cold room)
Wash with ~50mL NB
Elute with ~10mL NE, take 1mL fractions.
Concentrate as needed.
For all buffers see Sauer:Purification_of_His-tagged_proteins/Native_prep
Concentration can be determined with extinction of 19 770cm-1 at 280nm