Difference between revisions of "Team:Pasteur Paris/Silification"
| Line 28: | Line 28: | ||
color:#17A3B5; | color:#17A3B5; | ||
font-family: 'Oswald', Arial, sans-serif; | font-family: 'Oswald', Arial, sans-serif; | ||
| − | margin- | + | margin-right:50%; |
} | } | ||
| Line 37: | Line 37: | ||
color:#333; | color:#333; | ||
font-family: 'Oswald', Arial, sans-serif; | font-family: 'Oswald', Arial, sans-serif; | ||
| − | margin- | + | margin-left:5%; |
} | } | ||
h5 { | h5 { | ||
| Line 263: | Line 263: | ||
<h1><B>Cellulose binding notebook</B></h1> | <h1><B>Cellulose binding notebook</B></h1> | ||
| − | <div id="J1"><h2><B>August 24th, 2016 </B></h2></div> | + | <div id="J1"><h2><B>August 24th, 2016 </B></h2></div></br></br> |
| − | <h3> <strong>Cellulose binding test | + | <h3> <strong>Cellulose binding test </strong></h3> |
</div> | </div> | ||
<div class="text1"> | <div class="text1"> | ||
| Line 284: | Line 284: | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br> | + | 1.Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br> |
| − | In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), 100 µL of BSA solution and 10mg of Avicell.</br> | + | 2.In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), 100 µL of BSA solution and 10mg of Avicell.</br> |
| − | Let rocking 1h at room temperature</br> | + | 3.Let rocking 1h at room temperature</br> |
| − | Centrifuge 2min at 13.000 rpm</br> | + | 4.Centrifuge 2min at 13.000 rpm</br> |
| − | Take 1mL of the supernatent and store it in a 2mL eppendorf</br> | + | 5.Take 1mL of the supernatent and store it in a 2mL eppendorf</br> |
| − | Resuspend the pellet in 1mL of Buffer A</br> | + | 6.Resuspend the pellet in 1mL of Buffer A</br> |
| − | Vortex and let rocking during 2min at RT</br> | + | 7.Vortex and let rocking during 2min at RT</br> |
| − | Centrifuge 2min at 13.000rpm</br> | + | 8.Centrifuge 2min at 13.000rpm</br> |
| − | Collect the supernatent and pool it with the first taken</br> | + | 9.Collect the supernatent and pool it with the first taken</br> |
| − | Resuspend the pellet in 800 µL of Laemmli 2X</br> | + | 10.Resuspend the pellet in 800 µL of Laemmli 2X</br> |
| − | Denaturate the protein by heating them at 95°C during 5min</br> | + | 11.Denaturate the protein by heating them at 95°C during 5min</br> |
| − | Centrifuge 2min at 13.000rpm</br> | + | 12.Centrifuge 2min at 13.000rpm</br> |
| − | Depose 20 µL of the supernatent</br> | + | 13.Depose 20 µL of the supernatent</br> |
| − | For the other samples: mix 10 µL of Laemmli 2X with 10 µL of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br> | + | 14.For the other samples: mix 10 µL of Laemmli 2X with 10 µL of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br> |
Deposit table on the gel:</br> | Deposit table on the gel:</br> | ||
| − | + | Protein ruler</br> | |
| − | + | ///</br> | |
| − | + | Pellet</br> | |
| − | + | ///</br> | |
| − | + | Supernatant</br> | |
| − | + | ///</br> | |
| − | + | Prep2</br> | |
| + | ///</br> | ||
| + | BSA</br></br> | ||
Start at 10:15AM</br></br> | Start at 10:15AM</br></br> | ||
| Line 337: | Line 339: | ||
<U>Method:</U></br> This protocol is done three times with the different cellulose.</br> | <U>Method:</U></br> This protocol is done three times with the different cellulose.</br> | ||
| − | Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br> | + | 1.Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br> |
| − | In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), 100 µL of BSA solution and 10mg of Avicell.</br> | + | 2.In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), 100 µL of BSA solution and 10mg of Avicell.</br> |
| − | Let rocking 1h at room temperature</br> | + | 3.Let rocking 1h at room temperature</br> |
| − | Centrifuge 2min at 13.000 rpm</br> | + | 4.Centrifuge 2min at 13.000 rpm</br> |
| − | Take 1mL of the supernatent and store it in a 2mL eppendorf</br> | + | 5.Take 1mL of the supernatent and store it in a 2mL eppendorf</br> |
| − | Resuspend the pellet in 1mL of Buffer A</br> | + | 6.Resuspend the pellet in 1mL of Buffer A</br> |
| − | Vortex and let rocking during 2min at RT</br> | + | 7.Vortex and let rocking during 2min at RT</br> |
| − | Centrifuge 2min at 13.000rpm</br> | + | 8.Centrifuge 2min at 13.000rpm</br> |
| − | Collect the supernatent and pool it with the first taken</br> | + | 9.Collect the supernatent and pool it with the first taken</br> |
| − | Resuspend the pellet in 800 µL of Laemmli 2X</br> | + | 10.Resuspend the pellet in 800 µL of Laemmli 2X</br> |
| − | Denaturate the protein by heating them at 95°C during 5min</br> | + | 11.Denaturate the protein by heating them at 95°C during 5min</br> |
| − | Centrifuge 2min at 13.000rpm</br> | + | 12.Centrifuge 2min at 13.000rpm</br> |
| − | Depose 20 µL of the supernatent</br> | + | 13.Depose 20 µL of the supernatent</br> |
| − | For the other samples: mix 10 µL of Laemmli 2X with 10 µL of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br> | + | 14.For the other samples: mix 10 µL of Laemmli 2X with 10 µL of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br> |
Deposit table on the gel:</br> | Deposit table on the gel:</br> | ||
| − | + | Protein ruler</br> | |
| − | + | ///</br> | |
| − | + | Pellet (BSA + protein + Sigmacell)</br> | |
| − | + | Supernatant (BSA + protein + Sigmacell)</br> | |
| − | + | Pellet (BSA + Sigmacell)</br> | |
| − | + | Supernatant (BSA + Sigmacell)</br></br> | |
| − | + | Pellet (BSA + protein + Avicell)</br> | |
| − | + | Supernatant (BSA + protein + Avicell)</br></br> | |
| − | + | Pellet (BSA + protein + CMC)</br> | |
| − | + | Supernatant (BSA + protein + CMC)</br> | |
| − | + | Pellet (BSA + CMC)</br> | |
| − | + | Supernatant (BSA + CMC)</br></br> | |
| Line 404: | Line 406: | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br> | + | 1.Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br> |
| − | In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), | + | 2.In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), 100 µL of BSA solution and 10mg of Avicell.</br> |
| − | Let rocking 1h at room temperature</br> | + | 3.Let rocking 1h at room temperature</br> |
| − | Centrifuge 2min at 13.000 rpm</br> | + | 4.Centrifuge 2min at 13.000 rpm</br> |
| − | Take 1mL of the supernatent and store it in a 2mL eppendorf | + | 5.Take 1mL of the supernatent and store it in a 2mL eppendorf</br> |
| − | Resuspend the pellet in 1mL of Buffer A</br> | + | 6.Resuspend the pellet in 1mL of Buffer A</br> |
| − | Vortex and let rocking during 2min at RT</br> | + | 7.Vortex and let rocking during 2min at RT</br> |
| − | Centrifuge 2min at 13.000rpm</br> | + | 8.Centrifuge 2min at 13.000rpm</br> |
| − | Collect the supernatent | + | 9.Collect the supernatent and pool it with the first taken</br> |
| − | + | 10.Resuspend the pellet in 800 µL of Laemmli 2X</br> | |
| − | + | 11.Denaturate the protein by heating them at 95°C during 5min</br> | |
| − | + | 12.Centrifuge 2min at 13.000rpm</br> | |
| − | + | 13.Depose 20 µL of the supernatent</br> | |
| − | Resuspend the pellet in 800 µL of Laemmli 2X</br> | + | 14.For the other samples: mix 10 µL of Laemmli 2X with 10 µL of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br> |
| − | Denaturate the protein by heating them at 95°C during 5min</br> | + | |
| − | Centrifuge 2min at 13.000rpm</br> | + | |
| − | Depose 20 µL of | + | |
| − | For the other samples: mix 10 µL of Laemmli 2X with 10 µL of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br> | + | |
Deposit table on the gel:</br> | Deposit table on the gel:</br> | ||
| Line 469: | Line 468: | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br> | + | 1. Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br> |
| − | In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), 10 µL of BSA solution and 10mg of Avicell.</br> | + | 2. In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), 10 µL of BSA solution and 10mg of Avicell.</br> |
| − | Let rocking 1h at room temperature</br> | + | 3. Let rocking 1h at room temperature</br> |
| − | Centrifuge 2min at 13.000 rpm</br> | + | 4. Centrifuge 2min at 13.000 rpm</br> |
| − | Take 1mL of the supernatent and store it in a 2mL eppendorf (Supernatant 1)</br> | + | 5. Take 1mL of the supernatent and store it in a 2mL eppendorf (Supernatant 1)</br> |
| − | Resuspend the pellet in 1mL of Buffer A</br> | + | 6. Resuspend the pellet in 1mL of Buffer A</br> |
| − | Vortex and let rocking during 2min at RT</br> | + | 7. Vortex and let rocking during 2min at RT</br> |
| − | Centrifuge 2min at 13.000rpm</br> | + | 8. Centrifuge 2min at 13.000rpm</br> |
| − | Collect the supernatent in a clean eppendorf (Supernatant 2)</br> | + | 9. Collect the supernatent in a clean eppendorf (Supernatant 2)</br> |
| − | Resuspend the pellet in 1mL of Buffer A</br> | + | 10. Resuspend the pellet in 1mL of Buffer A</br> |
| − | Vortex and let rocking during 2min at RT</br> | + | 11. Vortex and let rocking during 2min at RT</br> |
| − | Centrifuge 2min at 13.000rpm</br> | + | 12. Centrifuge 2min at 13.000rpm</br> |
| − | Collect the supernatent in a clean eppendorf (Supernatant 3)</br> | + | 13. Collect the supernatent in a clean eppendorf (Supernatant 3)</br> |
| − | Resuspend the pellet in 200 µL of Laemmli 2X</br> | + | 14. Resuspend the pellet in 200 µL of Laemmli 2X</br> |
| − | Denaturate the protein by heating them at 95°C during 5min</br> | + | 15. Denaturate the protein by heating them at 95°C during 5min</br> |
| − | Centrifuge 2min at 13.000rpm</br> | + | 16. Centrifuge 2min at 13.000rpm</br> |
| − | Depose 20 µL of each supernatent</br> | + | 17. Depose 20 µL of each supernatent</br> |
| − | For the other samples: mix 10 µL of Laemmli 2X with 10 µL of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br> | + | 18. For the other samples: mix 10 µL of Laemmli 2X with 10 µL of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br> |
Deposit table on the gel:</br> | Deposit table on the gel:</br> | ||
| Line 501: | Line 500: | ||
///</br>
BSA</br></br> | ///</br>
BSA</br></br> | ||
| − | + | 19. Wash the gel three times with distilled water during 5min.</br> | |
| − | + | 20. Color the gel with Coomassie Blue diluted 1/5 during 30min.</br> | |
| − | + | 21. Wash with distilled water for 5min then let wash 15min.</br></br> | |
</p> | </p> | ||
| Line 534: | Line 533: | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br> | + | 1. Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br> |
| − | In a 1.5mL eppendorf, follow the next volume table:</br> | + | 2. In a 1.5mL eppendorf, follow the next volume table:</br> |
<table> | <table> | ||
<thead> | <thead> | ||
| Line 590: | Line 589: | ||
| − | Let rocking 2h at room temperature</br> | + | 3. Let rocking 2h at room temperature</br> |
| − | Centrifuge 2min at 13.000 rpm</br> | + | 4. Centrifuge 2min at 13.000 rpm</br> |
| − | Take 1mL of the supernatent and store it in a 2mL eppendorf (Supernatant 1)</br> | + | 5. Take 1mL of the supernatent and store it in a 2mL eppendorf (Supernatant 1)</br> |
| − | Resuspend the pellet in 1mL of Buffer A</br> | + | 6. Resuspend the pellet in 1mL of Buffer A</br> |
| − | Vortex and let rocking during 2min at RT</br> | + | 7. Vortex and let rocking during 2min at RT</br> |
| − | Centrifuge 2min at 13.000rpm</br> | + | 8. Centrifuge 2min at 13.000rpm</br> |
| − | Collect the supernatent in a clean eppendorf (Supernatant 2)</br> | + | 9. Collect the supernatent in a clean eppendorf (Supernatant 2)</br> |
| − | Resuspend the pellet in 200 µL of Laemmli 2X</br> | + | 10. Resuspend the pellet in 200 µL of Laemmli 2X</br> |
| − | Denaturate the protein by heating them at 95°C during 5min</br> | + | 11. Denaturate the protein by heating them at 95°C during 5min</br> |
| − | Centrifuge 2min at 13.000rpm</br> | + | 12. Centrifuge 2min at 13.000rpm</br> |
| − | Depose 40 µL of each supernatent</br> | + | 13. Depose 40 µL of each supernatent</br> |
| − | For the other samples: mix 20 µL of Laemmli 2X with 20 µL of protein (BSA alone, protein solution alone). Denaturate them 5min at 95°C</br></br> | + | 14. For the other samples: mix 20 µL of Laemmli 2X with 20 µL of protein (BSA alone, protein solution alone). Denaturate them 5min at 95°C</br></br> |
Deposit table on the gel:</br> | Deposit table on the gel:</br> | ||
| Line 648: | Line 647: | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br> | + | 1. Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br> |
| − | In a 1.5mL eppendorf, follow the next volume table:</br> | + | 2. In a 1.5mL eppendorf, follow the next volume table:</br> |
<table> | <table> | ||
| Line 713: | Line 712: | ||
| − | Let rocking 2h at room temperature</br> | + | 3. Let rocking 2h at room temperature</br> |
| − | Centrifuge 2min at 13.000 rpm</br> | + | 4. Centrifuge 2min at 13.000 rpm</br> |
| − | Take 1mL of the supernatent and store it in a 2mL eppendorf (Supernatant 1)</br> | + | 5. Take 1mL of the supernatent and store it in a 2mL eppendorf (Supernatant 1)</br> |
| − | Resuspend the pellet in 1mL of Buffer A</br> | + | 6. Resuspend the pellet in 1mL of Buffer A</br> |
| − | Vortex and let rocking during 2min at RT</br> | + | 7. Vortex and let rocking during 2min at RT</br> |
| − | Centrifuge 2min at 13.000rpm</br> | + | 8. Centrifuge 2min at 13.000rpm</br> |
| − | Collect the supernatent in a clean eppendorf (Supernatant 2)</br> | + | 9. Collect the supernatent in a clean eppendorf (Supernatant 2)</br> |
| − | Resuspend the pellet in 200 µL of Laemmli 2X</br> | + | 10. Resuspend the pellet in 200 µL of Laemmli 2X</br> |
| − | Denaturate the protein by heating them at 95°C during 5min</br> | + | 11. Denaturate the protein by heating them at 95°C during 5min</br> |
| − | Centrifuge 2min at 13.000rpm</br> | + | 12. Centrifuge 2min at 13.000rpm</br> |
| − | Depose 40 µL of each supernatent</br> | + | 13. Depose 40 µL of each supernatent</br> |
| − | For the other samples: mix 20 µL of Laemmli 2X with 20 µL of protein (BSA alone, protein solution alone). Denaturate them 5min at 95°C | + | 12. For the other samples: mix 20 µL of Laemmli 2X with 20 µL of protein (BSA alone, protein solution alone). Denaturate them 5min at 95°C |
</br></br> | </br></br> | ||
Deposit table on the gel:</br> | Deposit table on the gel:</br> | ||
