2.2 Material
for Western Blot
2.2.1 Cell Lysis
and Loading Buffer
1. 1 M Tris–HCl pH 8.0: in a beaker, dissolve 60.6 g of Tris base
in approximately 300 mL H 2 O. Add 37% HCl very slowly up
to pH ¼ 8.0 (see Note 4). Complete with H 2 O up to 500 mL
with a graduated cylinder.
2. Triton-X100.
3. 1 M dithiothreitol (DTT) in H 2 O.
4. 0.5 M EDTA pH 8.0: in a beaker, dissolve 146.1 g of EDTA
powder in 700 mL H 2 O. Add 10 M NaOH very slowly, up to
pH ¼ 8.0. Complete with H 2 O up to 1 L with a graduate
cylinder.
5. 0.2 M Sodium orthovanadate: in a beaker, dissolve 3.68 g of
sodium orthovanadate in 80 mL H 2 O. To dissolve the powder,
heat up the solution up to 50
C. Complete with H 2 O up to
100 mL with a graduate cylinder.
6. Sodium deoxycholate 10% (w/v) in H 2 O: For 100 mL, in a
beaker, dilute 10 g of sodium deoxycholate in 80 mL of
H 2 O. Complete with H 2 O up to 100 mL with a graduate
cylinder.
7. Protease inhibitor.
8. Lysis buffer: for 10 mL solution, combine 200 μL of 1 M Tris–
HCl, 10 μL of 1 M DTT, 20 μL of 0.5 M EDTA, 100 μL
Triton-X100, 500 μL of sodium deoxycholate 10% (w/v),
50 μL of 0.2 M sodium orthovanadate, ½ protease inhibitor
tablet and 9120 μL of H 2 O (see Note 5).
9. 4Â Laemmli sample buffer with β-mercaptoethanol: add
100 μL of 99% β-mercaptoethanol (see Note 6) to 900 μL of
4Â Laemmli sample buffer.
2.2.2 Casting of SDSPolyacrylamide Gels
1. Ammonium persulfate (APS) 10% (w/v) in H 2 O: for 100 mL,
in a beaker, dilute 10 g of APS in 80 mL of H 2 O. Complete
with H 2 O up to 100 mL with a graduate cylinder (see Note 7).
2. N,N,N,N
0 -Tetramethyl-ethylenediamine (TEMED) (see Note
8).
3. 3 M Tris–HCl pH 8.8: For 100 mL, in a beaker, dissolve
181.71 g of Tris base in approximately 450 mL H 2 O. Add
37% HCl very slowly until reaching pH ¼ 8.8 (see Note 4).
Complete with H 2 O up to 500 mL with a graduated cylinder.
4. 3 M Tris–HCl pH 6.8. For 100 mL: in a beaker, dissolve
36.34 g of Tris base in approximately 60 mL H 2 O. Add 37%
HCl very slowly up to pH ¼ 6.8 (see Note 4). Complete with
H 2 O to 100 mL with a graduated cylinder.
5. Resolving gel: 375 mM Tris–HCl pH 8.8, 10% Acrylamide:BisAcrylamide 29:1 (see Note 9), 0.1% SDS, 0.1% APS, 0.04%
TEMED. For one gel of 10 cm  7.5 cm  1 mm: mix 4.9 mL
H 2 O, 1 mL 3 M Tris pH 8.8, 2 mL Acrylamide:Bis-Acrylamide
356
Noe ´ mie Kempf et al.
for Western Blot
2.2.1 Cell Lysis
and Loading Buffer
1. 1 M Tris–HCl pH 8.0: in a beaker, dissolve 60.6 g of Tris base
in approximately 300 mL H 2 O. Add 37% HCl very slowly up
to pH ¼ 8.0 (see Note 4). Complete with H 2 O up to 500 mL
with a graduated cylinder.
2. Triton-X100.
3. 1 M dithiothreitol (DTT) in H 2 O.
4. 0.5 M EDTA pH 8.0: in a beaker, dissolve 146.1 g of EDTA
powder in 700 mL H 2 O. Add 10 M NaOH very slowly, up to
pH ¼ 8.0. Complete with H 2 O up to 1 L with a graduate
cylinder.
5. 0.2 M Sodium orthovanadate: in a beaker, dissolve 3.68 g of
sodium orthovanadate in 80 mL H 2 O. To dissolve the powder,
heat up the solution up to 50
C. Complete with H 2 O up to
100 mL with a graduate cylinder.
6. Sodium deoxycholate 10% (w/v) in H 2 O: For 100 mL, in a
beaker, dilute 10 g of sodium deoxycholate in 80 mL of
H 2 O. Complete with H 2 O up to 100 mL with a graduate
cylinder.
7. Protease inhibitor.
8. Lysis buffer: for 10 mL solution, combine 200 μL of 1 M Tris–
HCl, 10 μL of 1 M DTT, 20 μL of 0.5 M EDTA, 100 μL
Triton-X100, 500 μL of sodium deoxycholate 10% (w/v),
50 μL of 0.2 M sodium orthovanadate, ½ protease inhibitor
tablet and 9120 μL of H 2 O (see Note 5).
9. 4Â Laemmli sample buffer with β-mercaptoethanol: add
100 μL of 99% β-mercaptoethanol (see Note 6) to 900 μL of
4Â Laemmli sample buffer.
2.2.2 Casting of SDSPolyacrylamide Gels
1. Ammonium persulfate (APS) 10% (w/v) in H 2 O: for 100 mL,
in a beaker, dilute 10 g of APS in 80 mL of H 2 O. Complete
with H 2 O up to 100 mL with a graduate cylinder (see Note 7).
2. N,N,N,N
0 -Tetramethyl-ethylenediamine (TEMED) (see Note
8).
3. 3 M Tris–HCl pH 8.8: For 100 mL, in a beaker, dissolve
181.71 g of Tris base in approximately 450 mL H 2 O. Add
37% HCl very slowly until reaching pH ¼ 8.8 (see Note 4).
Complete with H 2 O up to 500 mL with a graduated cylinder.
4. 3 M Tris–HCl pH 6.8. For 100 mL: in a beaker, dissolve
36.34 g of Tris base in approximately 60 mL H 2 O. Add 37%
HCl very slowly up to pH ¼ 6.8 (see Note 4). Complete with
H 2 O to 100 mL with a graduated cylinder.
5. Resolving gel: 375 mM Tris–HCl pH 8.8, 10% Acrylamide:BisAcrylamide 29:1 (see Note 9), 0.1% SDS, 0.1% APS, 0.04%
TEMED. For one gel of 10 cm  7.5 cm  1 mm: mix 4.9 mL
H 2 O, 1 mL 3 M Tris pH 8.8, 2 mL Acrylamide:Bis-Acrylamide
356
Noe ´ mie Kempf et al.