Difference between revisions of "Team:Pasteur Paris/Binding cellulose"
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August 24th, 2016 August 25th, 2016 August 29th, 2016 September 12th, 2016 September 14th, 2016 September 29th, 2016
Aim: This step check the ability of our protein to bind cellulose. Moreover, this step will confirm the good design of the protein. Materials: • Cellulose Avicell (Sigma-Aldrich) • BSA Protein • Rocker • Centrifuge • Laemmli 2X • Buffer A (50mM Tris, 150mM of NaCl) • Heating table • SDS-Page gel and cuve • Protein ruler • Microbiology equipment Method: 1.Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A) 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. 3.Let rocking 1h at room temperature 4.Centrifuge 2min at 13.000 rpm 5.Take 1mL of the supernatent and store it in a 2mL eppendorf 6.Resuspend the pellet in 1mL of Buffer A 7.Vortex and let rocking during 2min at RT 8.Centrifuge 2min at 13.000rpm 9.Collect the supernatent and pool it with the first taken 10.Resuspend the pellet in 800 µL of Laemmli 2X 11.Denaturate the protein by heating them at 95°C during 5min 12.Centrifuge 2min at 13.000rpm 13.Depose 20 µL of the supernatent 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 Deposit table on the gel: Protein ruler /// Pellet /// Supernatant /// Prep2 /// BSA Start at 10:15AM 15. Wash the gel three times with distilled water during 5min. 16. Color the gel with Coomassie Blue diluted 1/5 during 30min. 17. Wash with distilled water for 5min then let wash 15min.
Aim: This step check the ability of our protein to bind cellulose. Moreover, this step will confirm the good design of the protein. Materials: • Cellulose Avicell (Sigma-Aldrich) • Cellulose Sigmacell (Sigma-Aldrich) • Carboxymethylcellulose (Sigma-Aldrich) • BSA Protein • Rocker • Centrifuge • Laemmli 2X • Buffer A (50mM Tris, 150mM of NaCl) • Heating table • SDS-Page gel and cuve • Protein ruler • Microbiology equipment Method: This protocol is done three times with the different cellulose. 1.Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A) 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. 3.Let rocking 1h at room temperature 4.Centrifuge 2min at 13.000 rpm 5.Take 1mL of the supernatent and store it in a 2mL eppendorf 6.Resuspend the pellet in 1mL of Buffer A 7.Vortex and let rocking during 2min at RT 8.Centrifuge 2min at 13.000rpm 9.Collect the supernatent and pool it with the first taken 10.Resuspend the pellet in 800 µL of Laemmli 2X 11.Denaturate the protein by heating them at 95°C during 5min 12.Centrifuge 2min at 13.000rpm 13.Depose 20 µL of the supernatent 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 Deposit table on the gel: Protein ruler /// Pellet (BSA + protein + Sigmacell) Supernatant (BSA + protein + Sigmacell) Pellet (BSA + Sigmacell) Supernatant (BSA + Sigmacell) Pellet (BSA + protein + Avicell) Supernatant (BSA + protein + Avicell) Pellet (BSA + protein + CMC) Supernatant (BSA + protein + CMC) Pellet (BSA + CMC) Supernatant (BSA + CMC) Start at 11:10AM at 130V. End at 12:20AM. 15. Wash the gel three times with distilled water during 5min. 16. Color the gel with Coomassie Blue diluted 1/5 during 30min. 17. Wash with distilled water for 5min then let wash 15min.
Aim: This step check the ability of our protein to bind cellulose. Moreover, this step will confirm the good design of the protein. Materials: • Cellulose Avicell (Sigma-Aldrich) • BSA Protein • Rocker • Centrifuge • Laemmli 2X • Buffer A (50mM Tris, 150mM of NaCl) • Heating table • SDS-Page gel and cuve • Protein ruler • Microbiology equipment Method: 1.Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A) 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. 3.Let rocking 1h at room temperature 4.Centrifuge 2min at 13.000 rpm 5.Take 1mL of the supernatent and store it in a 2mL eppendorf 6.Resuspend the pellet in 1mL of Buffer A 7.Vortex and let rocking during 2min at RT 8.Centrifuge 2min at 13.000rpm 9.Collect the supernatent and pool it with the first taken 10.Resuspend the pellet in 800 µL of Laemmli 2X 11.Denaturate the protein by heating them at 95°C during 5min 12.Centrifuge 2min at 13.000rpm 13.Depose 20 µL of the supernatent 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 Deposit table on the gel: Protein ruler /// Pellet /// Supernatant 1 /// Supernatant 2 /// Supernatant 3 /// Prep2 /// BSA 15. Wash the gel three times with distilled water during 5min. 16. Color the gel with Coomassie Blue diluted 1/5 during 30min. 17. Wash with distilled water for 5min then let wash 15min.
Aim: This step check the ability of our protein to bind cellulose. Moreover, this step will confirm the good design of the protein. Materials: • Cellulose Avicell (Sigma-Aldrich) • BSA Protein • Rocker • Centrifuge • Laemmli 2X • Buffer A (50mM Tris, 150mM of NaCl) • Heating table • SDS-Page gel and cuve • Protein ruler • Microbiology equipment Method: 1. Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A) 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. 3. Let rocking 1h at room temperature 4. Centrifuge 2min at 13.000 rpm 5. Take 1mL of the supernatent and store it in a 2mL eppendorf (Supernatant 1) 6. Resuspend the pellet in 1mL of Buffer A 7. Vortex and let rocking during 2min at RT 8. Centrifuge 2min at 13.000rpm 9. Collect the supernatent in a clean eppendorf (Supernatant 2) 10. Resuspend the pellet in 1mL of Buffer A 11. Vortex and let rocking during 2min at RT 12. Centrifuge 2min at 13.000rpm 13. Collect the supernatent in a clean eppendorf (Supernatant 3) 14. Resuspend the pellet in 200 µL of Laemmli 2X 15. Denaturate the protein by heating them at 95°C during 5min 16. Centrifuge 2min at 13.000rpm 17. Depose 20 µL of each supernatent 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 Deposit table on the gel: Protein ruler /// Pellet /// Supernatant 1 /// Supernatant 2 /// Supernatant 3 /// Prep2 /// BSA 19. Wash the gel three times with distilled water during 5min. 20. Color the gel with Coomassie Blue diluted 1/5 during 30min. 21. Wash with distilled water for 5min then let wash 15min.
Aim: This step check the ability of our protein to bind cellulose. Moreover, this step will confirm the good design of the protein. Materials: • Cellulose Avicell (Sigma-Aldrich) • BSA Protein • Rocker • Centrifuge • Laemmli 2X • Buffer A (50mM Tris, 150mM of NaCl) • Heating table • SDS-Page gel and cuve • Protein ruler • Microbiology equipment Method: 1. Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A) 2. In a 1.5mL eppendorf, follow the next volume table:
| Tube | 24kDa - sample 1 | 24kDa - sample 2 | 24kDa - sample 3 | 40kDa - sample 1 | 40kDa - sample 2 | 40kDa - sample 3 |
|---|---|---|---|---|---|---|
Protein solution |
500 µL | 250 µL | 500 µL | 500 µL | 250 µL | 500 µL |
BSA |
10 µL | 10 µL | 10 µL | 10 µL | 10 µL | 10 µL |
Avicell |
10 mg | 10 mg | 10 mg | 10 mg | 10 mg | 10 mg |
Buffer A |
500 µL | 750 µL | 500 µL | 500 µL | 750 µL | 500 µL |
Aim: This step check the ability of our protein to bind cellulose. Moreover, this step will confirm the good design of the protein purified on the 26/09. Materials: • Cellulose Avicell (Sigma-Aldrich) • BSA Protein • Rocker • Centrifuge • Laemmli 2X • Buffer A (50mM Tris, 150mM of NaCl) • Heating table • SDS-Page gel and cuve • Protein ruler • Microbiology equipment Method: 1. Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A) 2. In a 1.5mL eppendorf, follow the next volume table:
| Fractions | 14 | 15 | 16 | 19 | 20 | BSA control |
|---|---|---|---|---|---|---|
Concentration (ng/µL ) |
782 µL | 1086 µL | 1157 µL | 982 µL | 980 µL | 1000 µL |
Protein solution |
300 µL | 300 µL | 300 µL | 300 µL | 300 µL | 0 µL |
BSA |
10 µL | 10 µL | 10 µL | 10 µL | 10 µL | 300 µL |
Avicell |
10 mg | 10 mg | 10 mg | 10 mg | 10 mg | 10 mg |
Buffer A |
690 µL | 690 µL | 690 µL | 690 µL | 690 µL | 690 µL |
