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Team:Bordeaux/Results/Purification
From 2014.igem.org
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{{:Team:Bordeaux/Tete}} | {{:Team:Bordeaux/Tete}} | ||
| - | [[File:Bdx2014 ITCmethod.jpg]] | + | |
| + | Cells are lysed and the protein mixture is collected. The ELP is purified by ITC (inverse transition cycling) <br> | ||
| + | ---- | ||
| + | [[File:Bdx2014 ITCmethod.jpg|left]] | ||
| + | Method overview: | ||
| + | |||
| + | - 2ml of polyethylenimine is added to the lysate, the mixture is centrifuged at 14,000rpm for 15min at 4°C. | ||
| + | |||
| + | - The supernatant is saved and brought to room temperature. NaCl (1 to 3M) is added while homogenising the solution. | ||
| + | |||
| + | - The mixture is then centrifuged at 14,000 rpm for 15min at 37°C. | ||
| + | |||
| + | - Supernatent is discarded and pellet is resuspended with 2ml of cold PBS. | ||
| + | |||
| + | - First and second steps are repeated but NaCl 5M is added so the ELP precipitate. | ||
| + | |||
| + | - Third and fourth steps are repeated. | ||
| + | |||
| + | - A final centrifugation is performed at 4°C for 10min (14,000rpm) | ||
| + | |||
| + | - The solution is dialysed and the samples '''(pure protein)''' are lyophilized. | ||
| + | |||
| + | ---- | ||
| + | <br> | ||
| + | [[File:Bdx2014 ELPtt.jpg|350px]] | ||
| + | <br><br><br><br><br><br> | ||
{{:Team:Bordeaux/Pied}} | {{:Team:Bordeaux/Pied}} | ||
Latest revision as of 03:07, 18 October 2014
Cells are lysed and the protein mixture is collected. The ELP is purified by ITC (inverse transition cycling)
Method overview:
- 2ml of polyethylenimine is added to the lysate, the mixture is centrifuged at 14,000rpm for 15min at 4°C.
- The supernatant is saved and brought to room temperature. NaCl (1 to 3M) is added while homogenising the solution.
- The mixture is then centrifuged at 14,000 rpm for 15min at 37°C.
- Supernatent is discarded and pellet is resuspended with 2ml of cold PBS.
- First and second steps are repeated but NaCl 5M is added so the ELP precipitate.
- Third and fourth steps are repeated.
- A final centrifugation is performed at 4°C for 10min (14,000rpm)
- The solution is dialysed and the samples (pure protein) are lyophilized.
