Biology Reference
In-Depth Information
4. Acetone (80 % v/v) solution. Store at −20 °C and use directly
from the freezer.
5. Phenol solution equilibrated with 10 mM Tris-HCl, pH 8
(Sigma, Chemical Company). Store at 4 °C.
6. Sodium dodecyl sulfate (SDS) buffer: 0.1 M Tris-HCl, pH 8,
30 % (w/v) sucrose, 2 % (w/v) SDS, 5 % (v/v)
β
-mercaptoethanol. Store at 4 °C and temper prior to use. Add
the
-mercaptoethanol just before use.
7. Solubilization solution: 9 M urea, 2 M thiourea, 4 % (w/v)
3-[(3-cholamidopropyl) dimethylammonio]-1-
propanesulfonate (CHAPS), 0.5 % (v/v) Tritón-X100, 20 mM
dithiothreitol (DTT). Store in 1 mL aliquots at −20 °C. Add
the DTT just before use.
β
1. Bradford solution (Sigma, Chemical Company) (
see
Note 1
).
Store at 4 °C.
2. Protein standard: bovine serum albumin (BSA) at a concentra-
tion of 1 mg/mL in distilled water is used as a stock solution
(
see
Note 2
). Store at −20 °C.
2.1.2 Protein
Quantifi cation
1. 1.5 M Tris-HCl, pH 8.8. Store at 4 °C.
2. 0.5 M Tris-HCl, pH 6.8. Store at 4 °C.
3. 30 % Acrylamide/Bisacrylamide solution, 37.5:1 (Bio-Rad).
Store at 4 °C.
4. Sodium dodecyl sulfate solution (SDS): 10 % (w/v) in water.
Store at room temperature.
5. Ammonium persulfate solution (APS): 10 % (w/v) in water.
Store at −20 °C (
see
Note 3
).
6.
N
,
N
,
N
2.1.3 One-Dimensional
Gel Electrophoresis
-Tetramethylethylenediamine (TEMED) (Sigma)
(
see
Note 4
). Store at room temperature.
7. Running buffer: 25 mM Tris-HCl, pH 8.3, 192 mM glycine,
0.1 % (w/v) SDS. Store at room temperature (
see
Note 5
).
8. Laemmli buffer: 62.5 mM Tris-HCl, pH 6.8, 25 % (v/v) glyc-
erol, 2 % (w/v) SDS, 0.01 % (w/v) bromophenol blue, 5 %
(v/v)
′
,
N
′
β
-mercaptoethanol. Store at room temperature. Add the
-mercaptoethanol just before use.
9. Standard broad range molecular weight markers (Bio-Rad).
Store at 4 °C.
β
1. Immobilized pH gradient strips of pH range 5-10, and 17 cm
length (Bio-Rad).
2. Rehydration solution: 7 M urea, 2 M thiourea, 4 % (w/v)
CHAPS, 0.5 % (v/v) anfolites, 20 mM DTT, and 0.01 %
(w/v) bromophenol blue. Store in 1 mL aliquots at −20 °C.
Add the DTT just before use.
2.1.4 Two-Dimensional
Gel Electrophoresis
