Difference between revisions of "Team:Pasteur Paris/Microbiology week17"
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<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | Melt the pellet of bacteria C2 (from | + | Melt the pellet of bacteria C2 (from 1 l culture) and resuspend it with 10 ml of buffer A. We had 25 ml of pellet we complete until 40 ml with buffer A.</br> |
| − | Add 40 µ | + | Add 40 µl of PMSF to avoid protein denaturation.</br> |
Put the column on the FPLC, clean the FPLC with water and then fill it with buffer A.</br> | Put the column on the FPLC, clean the FPLC with water and then fill it with buffer A.</br> | ||
| − | Sonicate the sample three times one minute at 60 | + | Sonicate the sample three times one minute at 60 %, wait 90 seconds between each sonication.</br> |
Centrifuge 25 min at 16000g (rotor JA 25.50)</br> | Centrifuge 25 min at 16000g (rotor JA 25.50)</br> | ||
Inject your sample in the FPLC</br> | Inject your sample in the FPLC</br> | ||
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• SN: Supernatant</br> | • SN: Supernatant</br> | ||
• F: Flow through (unfixed proteins)</br> | • F: Flow through (unfixed proteins)</br> | ||
| − | • W: Wash 5 | + | • W: Wash 5 % of buffer B </br> |
• Fractions (depending on the gradient)</br></br> | • Fractions (depending on the gradient)</br></br> | ||
