2.1 Purification of
Recombinant Proteins
1. Plasmids: rat Pdx1-pET21a, rat NeuroD-pET21a, and mouse
MafA-11R-pGEX-6p-1 (see Note 1).
2. 0.1 M Isopropyl-β-D-thiogalactopyranoside (IPTG): 0.1 M in
water. Store at À20
C.
3. Lysis buffer for histidine (His)-tag purification: 20 mM
HEPES-NaOH (pH 8.0), 100 mM NaCl, 1% Triton X-100,
and 0.1 mg/mL lysozyme. Store at 4
C. Add 1 tablet/50 mL
cOmplete EDTA-free protease inhibitor cocktail (Roche)
before use.
4. Wash buffer for His-tag purification: 20 mM HEPES-NaOH
(pH 8.0), 100 mM NaCl, and 20 mM imidazole. Store at 4
C.
Add 1 tablet/50 mL cOmplete EDTA-free protease inhibitor
cocktail (Roche) before use.
5. Elution buffer for His-tag purification: 20 mM HEPES-NaOH
(pH 8.0) 100 mM NaCl, and 500 mM imidazole. Store at
4
C. Add 1 tablet/50 mL cOmplete EDTA-free protease
inhibitor cocktail (Roche) before use.
6. Lysis buffer 1 for glutathione-S-transferase (GST)-tag purification: 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 1 mM
EDTA, and 1 mM EGTA. Store at 4
C.
7. Lysis buffer 2 for GST-tag purification: 50 mM Tris–HCl
(pH 8.0), 300 mM NaCl, 1 mM EDTA, 1 mM EGTA, 4 mM
DTT, 0.8% NP40, and 2 mg/mL lysozyme. Store at 4
C. Add
1 tablet/25 mL cOmplete protease inhibitor cocktail (Roche)
before use.
8. Wash buffer for GST-tag purification: 20 mM Tris–HCl
(pH 8.0), 2 mM MgCl 2 , and 200 mM NaCl. Store at 4
C.
9. GST cleavage buffer: 50 mM Tris–HCl (pH 7.0), 150 mM
NaCl, 1 mM EDTA, and 1 mM DTT. Store at 4
C.
2.2 Culture of
Embryonic Stem Cells
Make all media, buffers, and solutions sterile.
1. 0.1% Gelatin solution: Add 100 mL of water to a 100 mL
medium bottle. Weigh 0.1 g of gelatin and transfer to the
bottle. Mix and leave to stand for 30 min. Sterilize with an
autoclave. Store at 4
C.
2. Mouse embryonic fibroblast (MEF) medium: high-glucose
(4500 mg/L) DMEM supplemented with 10% fetal bovine
serum (FBS), 2 mM L-glutamine (L-Gln), 50 U/mL penicillin,
and 50 μg/mL streptomycin. Store at 4
C.
3. Embryonic stem cell (ESC) medium: Glasgow MEM supplemented with 1000 U/mL leukemia inhibitory factor (LIF),
15% knockout serum replacement (KSR; Thermo Fisher Scientific), 1% FBS, 100 μM nonessential amino acids (NEAAs),
Pancreatic Differentiation Through Protein Transduction
87
Recombinant Proteins
1. Plasmids: rat Pdx1-pET21a, rat NeuroD-pET21a, and mouse
MafA-11R-pGEX-6p-1 (see Note 1).
2. 0.1 M Isopropyl-β-D-thiogalactopyranoside (IPTG): 0.1 M in
water. Store at À20
C.
3. Lysis buffer for histidine (His)-tag purification: 20 mM
HEPES-NaOH (pH 8.0), 100 mM NaCl, 1% Triton X-100,
and 0.1 mg/mL lysozyme. Store at 4
C. Add 1 tablet/50 mL
cOmplete EDTA-free protease inhibitor cocktail (Roche)
before use.
4. Wash buffer for His-tag purification: 20 mM HEPES-NaOH
(pH 8.0), 100 mM NaCl, and 20 mM imidazole. Store at 4
C.
Add 1 tablet/50 mL cOmplete EDTA-free protease inhibitor
cocktail (Roche) before use.
5. Elution buffer for His-tag purification: 20 mM HEPES-NaOH
(pH 8.0) 100 mM NaCl, and 500 mM imidazole. Store at
4
C. Add 1 tablet/50 mL cOmplete EDTA-free protease
inhibitor cocktail (Roche) before use.
6. Lysis buffer 1 for glutathione-S-transferase (GST)-tag purification: 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 1 mM
EDTA, and 1 mM EGTA. Store at 4
C.
7. Lysis buffer 2 for GST-tag purification: 50 mM Tris–HCl
(pH 8.0), 300 mM NaCl, 1 mM EDTA, 1 mM EGTA, 4 mM
DTT, 0.8% NP40, and 2 mg/mL lysozyme. Store at 4
C. Add
1 tablet/25 mL cOmplete protease inhibitor cocktail (Roche)
before use.
8. Wash buffer for GST-tag purification: 20 mM Tris–HCl
(pH 8.0), 2 mM MgCl 2 , and 200 mM NaCl. Store at 4
C.
9. GST cleavage buffer: 50 mM Tris–HCl (pH 7.0), 150 mM
NaCl, 1 mM EDTA, and 1 mM DTT. Store at 4
C.
2.2 Culture of
Embryonic Stem Cells
Make all media, buffers, and solutions sterile.
1. 0.1% Gelatin solution: Add 100 mL of water to a 100 mL
medium bottle. Weigh 0.1 g of gelatin and transfer to the
bottle. Mix and leave to stand for 30 min. Sterilize with an
autoclave. Store at 4
C.
2. Mouse embryonic fibroblast (MEF) medium: high-glucose
(4500 mg/L) DMEM supplemented with 10% fetal bovine
serum (FBS), 2 mM L-glutamine (L-Gln), 50 U/mL penicillin,
and 50 μg/mL streptomycin. Store at 4
C.
3. Embryonic stem cell (ESC) medium: Glasgow MEM supplemented with 1000 U/mL leukemia inhibitory factor (LIF),
15% knockout serum replacement (KSR; Thermo Fisher Scientific), 1% FBS, 100 μM nonessential amino acids (NEAAs),
Pancreatic Differentiation Through Protein Transduction
87