GST-fusion protein purification
Inoculate 5 ml of the GST-fusion protein expressing clone and grow ON.
Inoculate 100 ml with the ON culture and grow until OD600 = 0.4-0.5.
Induce with 500 μM IPTG and incubate 3h at 37°C or 5h at 25°C or ON at 16°C.
Centrifuge, wash with ice-cold PBS, and ressuspend in 5 ml of ice-cold lysis buffer.
Sonicate on ice 4X 15 sec at 40% (verify lysis under the microscope)
Centrifuge 30 min at 10000 rpm and 4°C (save pellet and SNT samples for SDS-PAGE)
Incubate the SNT with 250 μl of Glutathion Sepharose 4B beads (previously equilibrated in lysis buffer) 1h at 4°C.
Load the beads into a column and allow the SNT to flow through by gravity (save a sample of the FT for SDS-PAGE)
Wash 3X with ice cold PBS (save samples of each wash for SDS-PAGE)
Elute 1X with ½ beads volume of elution buffer and 3X with 1 volume (save samples of each elution and beads for SDS-PAGE)
Lysis buffer: 1% triton X100
2 mM DTT
PIC
in PBS
Elution buffer: 50 mM Tris pH 8.0
10 mM reduced glutathione
2 mM DTT
PIC
Inoculate 100 ml with the ON culture and grow until OD600 = 0.4-0.5.
Induce with 500 μM IPTG and incubate 3h at 37°C or 5h at 25°C or ON at 16°C.
Centrifuge, wash with ice-cold PBS, and ressuspend in 5 ml of ice-cold lysis buffer.
Sonicate on ice 4X 15 sec at 40% (verify lysis under the microscope)
Centrifuge 30 min at 10000 rpm and 4°C (save pellet and SNT samples for SDS-PAGE)
Incubate the SNT with 250 μl of Glutathion Sepharose 4B beads (previously equilibrated in lysis buffer) 1h at 4°C.
Load the beads into a column and allow the SNT to flow through by gravity (save a sample of the FT for SDS-PAGE)
Wash 3X with ice cold PBS (save samples of each wash for SDS-PAGE)
Elute 1X with ½ beads volume of elution buffer and 3X with 1 volume (save samples of each elution and beads for SDS-PAGE)
Lysis buffer: 1% triton X100
2 mM DTT
PIC
in PBS
Elution buffer: 50 mM Tris pH 8.0
10 mM reduced glutathione
2 mM DTT
PIC