Difference between revisions of "Team:Pasteur Paris/Microbiology week16"
| Line 246: | Line 246: | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
| − | • Fast | + | • Fast Protein Liquid Chromatography</br> |
| − | • Chaotropic reagent (Guanidinium 6M)</br> | + | • Chaotropic reagent (Guanidinium HCL 6M)</br> |
| − | • EDTA 0 | + | • EDTA 0.1 M</br> |
| − | • PMSF ( | + | • PMSF (100 mM)</br> |
• Ni<sup>2+</sup> solution (100mM)</br> | • Ni<sup>2+</sup> solution (100mM)</br> | ||
• Centrifuge </br> | • Centrifuge </br> | ||
| − | • Buffer A ( | + | • Buffer A ( Tris 50 mM pH 7.4, NaCl 150 mM)</br> |
| − | • Buffer B ( | + | • Buffer B ( Tris 50 mM pH 7.4, NaCl 150 mM, Imidazole 250 mM)</br></br> |
<U>Method:</U></br> | <U>Method:</U></br> | ||
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> | 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 µl of PMSF to avoid protein denaturation.</br> | + | Add 40 µl of PMSF 100 mM 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 %, wait 90 seconds between each sonication.</br> | Sonicate the sample three times one minute at 60 %, wait 90 seconds between each sonication.</br> | ||
