Sauer:GFP-H6-ssrA purification: Difference between revisions
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(New page: Expression 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 ...) |
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Expression | Expression | ||
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Centrifuge- 30m x 15krpm | Centrifuge- 30m x 15krpm | ||
Ni-NTA column | |||
pre-incubate supernatant with washed Ni-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 His-6 protein purification | |||
Revision as of 08:25, 18 June 2008
Expression
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) with 5mL H6 protein lysis buffer.
Purification 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 Ni-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 His-6 protein purification
