Team:Freiburg/Protocols/Overexpression
Protein overexpression and purification in E. coli
His-Tag; Strep-Tag protein purification
remarks concerning the protocol (author, adapted from etc.)
material: peptone, NaCl, yeast extract, IPTG , …
duration: … min
Protein overexpression
- o/n culture is inoculated 1:100 in LB-medium (corresponding antibiotics 1:1000)
- cells grow at 37°C until OD600 ~ 0.5
- after that cell culture is induced with a certain IPTG (Isopropyl-β-D-1-thiogalactopyranosid)
- cells were set to a specific temperature for a specific time
- the culture is harvested by centrifugation (20min, 5000 g, 4°C)
- discard the supernatant and freeze the pellet at -20°C
| Medium and buffers | |
|---|---|
| LB | 10 g peptone (w/v) [or 10 g Bacto-tryptone?] |
| 5 g NaCl [or 10 g ?] | |
| 5 g yeast extract | |
| add 1 l H2O; autoclave | |
| standardbuffer | 20 mM Tris pH 7.5 |
| 150 mM NaCl | |
| 0.3 mM PMSF | |
| 10mM Imidazole | |
| washing buffer | 20 mM Tris pH 7.5 |
| 150 mM NaCl | |
| 20 mM Imidazole | |
| elution buffer | 20 mM Tris pH 7.5 |
| 150 mM NaCl | |
| 500 mM Imidazole | |
