Team:Pasteur Paris/Binding cellulose

Cellulose binding notebook

August 24th, 2016



Cellulose binding test

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.

August 25th, 2016:



Cellulose binding test

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.




August 29th, 2016:



Cellulose binding test

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.




September 12th, 2016:



Cellulose binding test

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.




September 14th, 2016:



Cellulose binding test

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
Volumes



3. Let rocking 2h 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 200 µL of Laemmli 2X
11. Denaturate the protein by heating them at 95°C during 5min
12. Centrifuge 2min at 13.000rpm
13. Depose 40 µL of each supernatent
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

Deposit table on the gel:
BSA (x2)
///

Supernatant 1 (x6)
///
Supernatant 2 (x6)
///
Protein 24kDa
///
Protein 40kDa
///

Pellet (x6)

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.




September 29th, 2016:



Cellulose binding test

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
Volumes



3. Let rocking 2h 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 200 µL of Laemmli 2X
11. Denaturate the protein by heating them at 95°C during 5min
12. Centrifuge 2min at 13.000rpm
13. Depose 40 µL of each supernatent
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

Deposit table on the gel:
Fraction 14
///

Fraction 15
///
Fraction 16
///
Fraction 19
///
Fraction 20
///

BSA control

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.