Aim: This step checks the ability of our protein to catalyse silification. Moreover, this step will confirm the good design of the protein. Materials: • HCl • TEOS • 15mL Falcons Method: Make one test with two control tubes 1mL of HCl + 1mL of TEOS, 4min at RT then add 100 µL of protein solution Prep 2 after dyalisis and 8mL of Buffer A 1mL of HCl + 8mL of Buffer A , 4min at RT then add 100 µL of the protein solution (no silicic acid is produced) 1mL of HCl + 1mL of TEOS, 4min at RT then add 8mL of Buffer A (no protein control) Start at 8:45PM
Aim: This step checks the ability of our protein to catalyse silification. Moreover, this step will confirm the good design of the protein. We do this silification test with the band at 24kDa and at 40kDa. Materials: • HCl (1M) • TEOS • 15mL Falcons Method: Make one test with two control tubes 1mL of HCl + 1mL of TEOS, vortex 4min at RT then add 100 µL of protein solution and 3 mL of Buffer A 1mL of HCl + 3mL of Buffer A , 4min at RT then add 100 µL of the protein solution (no silicic acid is produced) 1mL of HCl + 1mL of TEOS, 4min at RT then add 3mL of Buffer A (no protein control)
Aim: This step checks the ability of our protein to catalyse silification. Moreover, this step will confirm the good design of the protein. We do this silification test with the band at 24kDa and at 40kDa. Materials: • HCl (1M) • TEOS • 15mL Falcons Method: Make one test with two control tubes 1mL of HCl + 1mL of TEOS, vortex 4min at RT then add 300 µL of protein solution and 1 mL of Buffer A 1mL of HCl + 1 mL of Buffer A , 4min at RT then add 300 µL of the protein solution (no silicic acid is produced) 1mL of HCl + 1mL of TEOS, 4min at RT then add 1 mL of Buffer A (no protein control) Stir the samples 2h30 at RT. Centrifuge 15min at 16100g. Results : We notice a pellet in the four tubes, it may be the precipitation of the protein itself.
Aim: This step checks the ability of our protein to catalyse silification. Moreover, this step will confirm the good design of the protein. We do this silification test with the band at 24kDa and at 40kDa. Materials: • HCl (1M) • TEOS • 15mL Falcons Method: Make one test with two control tubes. Do this experiment for fraction 14/15/16/19/20 and BSA control. 1mL of HCl + 22.4 µL of TEOS, vortex 4min at RT then add 300 µL of protein solution and 1 mL of Buffer A 1mL of HCl + 1 mL of Buffer A , 4min at RT then add 300 µL of the protein solution (no silicic acid is produced) 1mL of HCl + 1mL of TEOS, 4min at RT then add 1 mL of Buffer A (no protein control) Stir the samples 2h30 at RT. Centrifuge 15min at 16100g. Results : We notice a pellet in all tubes except in the controls.
