8. Wash 2.66 mL of Glutathione Sepharose 4 Fast Flow
(GE) beads with 20 mL of water. Centrifuge at 500 Â g for
2 min at 4
C and then remove the supernatant.
9. Equilibrate the beads with 20 mL of wash buffer. Centrifuge at
500 Â g for 2 min at 4
C and then remove the supernatant.
10. Apply the supernatant of the bacterial lysate to the equilibrated
beads and incubate them with gentle rotation for 1–2 h at 4
C.
11. Wash the beads with 20 mL of cold wash buffer three times.
12. Transfer the beads to new 15 mL conical tubes. Equilibrate
them with 10 mL of cold GST cleavage buffer. Centrifuge at
500 Â g for 2 min at 4
C and then remove the supernatant.
13. Prepare the PreScission protease (GE) mixture (see Note 10).
Mix 60 μL of PreScission protease and 1940 μL of GST cleavage buffer.
14. Add 2 mL of the PreScission protease mixture to the beads and
then rotate them for 4 h at 4
C to cleave the GST-tag (see Note
11).
15. Centrifuge the beads at 500 Â g for 5 min at 4
C and then
collect the supernatant into a new tube.
16. Elute the residual proteins with 6 mL of cold GST cleavage
buffer. Centrifuge the beads at 500 Â g for 5 min at 4
C and
then collect the supernatant.
17. Assess the yield and purity of the eluted proteins by SDS-PAGE
followed by CBB staining.
18. Dialyze the protein by 4 L of PBS for 2 h at 4
C and repeat
twice (see Note 9). Concentrate the protein until the concentration reaches 20 μM by centrifugal filter units. Sterilize
through a 0.22-μm filter. Store at À80
C.
3.3 Preparation of
Feeder Cells
1. Coat a culture dish with a 0.1% gelatin solution (2 mL/60 mm
dish). Incubate for 30 min at 37
C in a CO 2 incubator (see
Note 12).
2. Thaw mitomycin C-treated MEFs (see Note 13) quickly at
37
C in a water bath and transfer them to 15 mL conical
tubes containing 5 mL of MEF medium. Centrifuge at
200 Â g for 5 min and aspirate the medium. Resuspend the
cells with 4 mL of MEF medium and plate them into gelatincoated dishes at a density of 2~5 Â 10
5 cells/60 mm dish.
3. Culture the cells for 24 h at 37
C in a CO 2 incubator.
3.4 Culture of
Embryonic Stem Cells
1. Thaw the ESCs (see Note 14) quickly at 37
C in a water bath
and transfer them to 15 mL conical tubes containing 5 mL of
ESC medium. Centrifuge at 200 Â g for 5 min and then
Pancreatic Differentiation Through Protein Transduction
91
(GE) beads with 20 mL of water. Centrifuge at 500 Â g for
2 min at 4
C and then remove the supernatant.
9. Equilibrate the beads with 20 mL of wash buffer. Centrifuge at
500 Â g for 2 min at 4
C and then remove the supernatant.
10. Apply the supernatant of the bacterial lysate to the equilibrated
beads and incubate them with gentle rotation for 1–2 h at 4
C.
11. Wash the beads with 20 mL of cold wash buffer three times.
12. Transfer the beads to new 15 mL conical tubes. Equilibrate
them with 10 mL of cold GST cleavage buffer. Centrifuge at
500 Â g for 2 min at 4
C and then remove the supernatant.
13. Prepare the PreScission protease (GE) mixture (see Note 10).
Mix 60 μL of PreScission protease and 1940 μL of GST cleavage buffer.
14. Add 2 mL of the PreScission protease mixture to the beads and
then rotate them for 4 h at 4
C to cleave the GST-tag (see Note
11).
15. Centrifuge the beads at 500 Â g for 5 min at 4
C and then
collect the supernatant into a new tube.
16. Elute the residual proteins with 6 mL of cold GST cleavage
buffer. Centrifuge the beads at 500 Â g for 5 min at 4
C and
then collect the supernatant.
17. Assess the yield and purity of the eluted proteins by SDS-PAGE
followed by CBB staining.
18. Dialyze the protein by 4 L of PBS for 2 h at 4
C and repeat
twice (see Note 9). Concentrate the protein until the concentration reaches 20 μM by centrifugal filter units. Sterilize
through a 0.22-μm filter. Store at À80
C.
3.3 Preparation of
Feeder Cells
1. Coat a culture dish with a 0.1% gelatin solution (2 mL/60 mm
dish). Incubate for 30 min at 37
C in a CO 2 incubator (see
Note 12).
2. Thaw mitomycin C-treated MEFs (see Note 13) quickly at
37
C in a water bath and transfer them to 15 mL conical
tubes containing 5 mL of MEF medium. Centrifuge at
200 Â g for 5 min and aspirate the medium. Resuspend the
cells with 4 mL of MEF medium and plate them into gelatincoated dishes at a density of 2~5 Â 10
5 cells/60 mm dish.
3. Culture the cells for 24 h at 37
C in a CO 2 incubator.
3.4 Culture of
Embryonic Stem Cells
1. Thaw the ESCs (see Note 14) quickly at 37
C in a water bath
and transfer them to 15 mL conical tubes containing 5 mL of
ESC medium. Centrifuge at 200 Â g for 5 min and then
Pancreatic Differentiation Through Protein Transduction
91