Difference between revisions of "Team:Pasteur Paris/Silification"

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.

August 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 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:

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:

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:

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
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

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.

@@ Line 299: / Line 299: @@
 .For the other samples: mix 10 &#181;L of Laemmli 2X with 10 &#181;L of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br>
-Deposit table on the gel:</br>
+<U>Deposit table on the gel:</U></br>
 Protein ruler</br>
 ///</br>
@@ Line 354: / Line 354: @@
 .For the other samples: mix 10 &#181;L of Laemmli 2X with 10 &#181;L of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br>
-Deposit table on the gel:</br>
+<U>Deposit table on the gel:</U></br>
 Protein ruler</br>
 ///</br>
@@ Line 390: / Line 390: @@
                 <U> Aim:</U> This step check the ability of our protein to bind cellulose.
-Moreover, this step will confirm the good design of the protein.</br>
+Moreover, this step will confirm the good design of the protein.</br></br>
                 <U>Materials:</U></br>
@@ Line 422: / Line 422: @@
-Deposit table on the gel:</br>
+<U>Deposit table on the gel:</U></br>
 Protein ruler</br>
 ///</br>
@@ Line 487: / Line 487: @@
 . For the other samples: mix 10 &#181;L of Laemmli 2X with 10 &#181;L of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br>
-Deposit table on the gel:</br>
+<U>Deposit table on the gel:</U></br>
 Protein ruler</br>
 ///</br>
@@ Line 602: / Line 602: @@
 . For the other samples: mix 20 &#181;L of Laemmli 2X with 20 &#181;L of protein (BSA alone, protein solution alone). Denaturate them 5min at 95°C</br></br>
-Deposit table on the gel:</br>
+<U>Deposit table on the gel:</U></br>
 BSA (x2)</br>
 ///</br> Supernatant 1 (x6)</br>
@@ Line 725: / Line 725: @@
 . For the other samples: mix 20 &#181;L of Laemmli 2X with 20 &#181;L of protein (BSA alone, protein solution alone). Denaturate them 5min at 95°C
 </br></br>
-Deposit table on the gel:</br>
+<U>Deposit table on the gel:</U></br>
 Fraction 14</br>
 ///</br> Fraction 15</br>

Difference between revisions of "Team:Pasteur Paris/Silification"

Revision as of 21:00, 15 October 2016

Cellulose binding notebook

August 24th, 2016

Cellulose binding test

August 25th, 2016:

Cellulose binding test

August 29th, 2016:

Cellulose binding test

September 12th, 2016:

Cellulose binding test

September 14th, 2016:

Cellulose binding test

September 29th, 2016:

Cellulose binding test