Sauer:GFP-H6-ssrA purification: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(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 ...) |
No edit summary |
||
| (7 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
=== Expression === | |||
Grow cells at 30°C. | |||
Innoculate 1L of LB or TB + Amp with 10mL of overnight culture. | Innoculate 1L of LB or TB + Amp with 10mL of overnight culture. | ||
Induce cells with 0.5mM IPTG at OD ~0.7 | Induce cells with 0.5mM IPTG at OD ~0.7 | ||
After ~ 3 hrs harvest cells by centrifugation, 20m x 4krpm | After ~ 3 hrs harvest cells by centrifugation, 20m x 4krpm | ||
Purification | Freeze cells (-80°C) with 5mL H6 protein lysis buffer. | ||
=== Purification === | |||
Thaw with additional 5mL lysis buffer | Thaw with additional 5mL lysis buffer | ||
and protease inhibitor (Roche EDTA free protease inhibitor capsules work well) | and protease inhibitor (Roche EDTA free protease inhibitor capsules work well) | ||
| Line 15: | Line 18: | ||
Centrifuge- 30m x 15krpm | Centrifuge- 30m x 15krpm | ||
Ni-NTA column | |||
pre-incubate supernatant with washed Ni<sup>2+</sup>-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<sup>-1</sup> at 280nm | |||
Latest revision as of 08:32, 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°C) 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 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
