Biology Reference
In-Depth Information
water. Adjust pH with HCl (
see
Note 2
). Make up to 100 mL
with water in a graduated cylinder. Store at 4 °C.
2. Stacking gel buffer: 1 M Tris-HCl, pH 6.8. Weigh 12.1 g of
Tris-HCl and dissolve in about 80 mL of water. Adjust pH with
HCl (
see
Note 2
). Make up to 100 mL with water. Store at 4 °C.
3. 30 % (w/v) acrylamide/bisacrylamide mixture (30 % AA/BIS):
29 % (w/v) acrylamide, 1 % (w/v)
N
,
N
-methylenebisacrylamide.
Weigh 29.0 g of acrylamide and 1.0 g of bisacrylamide into a
glass beaker (
see
Note 5
) and dissolve in about 80 mL of water.
Make up to 100 mL with water in a graduated 100 mL
cylinder. Store at 4 °C (
see
Note 6
).
4. Sodium dodecyl sulfate (SDS) solution: 10 % (w/v) SDS in
water. Dissolve 20 mg of SDS in 200
′
μ
L of water. Prepare fresh
solution each time.
5. Ammonium persulfate (APS): 10 % (w/v) APS in water.
Dissolve 20 mg of APS in 200
μ
L of water. Prepare fresh solu-
tion each time.
6.
N
,
N
,
N
-tetramethylethylenediamine (TEMED).
7. SDS-PAGE running buffer, 10× concentrated: 0.25 M Tris-
HCl, 1.92 M glycine, 1 % (w/v) SDS. Weigh 15.1 g of Tris-
HCl, 72.1 g of glycine, and 5 g of SDS and make up to 500 mL
with water (
see
Note 7
). Do not adjust pH. Store at 4 °C.
Before use, dilute ten times.
8. Protein loading buffer (PLB): 0.1 M Tris-HCl, pH 6.8, 1.2 %
(w/v) SDS, 30 % (v/v) glycerol, 0.15 M dithiothreitol (DTT),
0.025 % bromophenol blue: Weigh 0.12 g of SDS, 0.23 g of
DTT, and 2.5 mg of bromophenol blue. Add 1 mL of stacking
gel buffer and 3 mL of glycerol (to measure viscous glycerol
proceed similarly as with Tween 20 in
Note 3
). Make up to
10 mL with water. Divide the solution in ten aliquots and store
at −20 °C.
9. Protein standards for SDS-PAGE.
10. Coomassie Brilliant Blue (CBB) G-250 solution: 0.4 % CBB
G-250 in 96 % ethanol. Weigh 0.4 g of CBB G-250 and dis-
solve in 100 mL of 96 % ethanol [
9
]. Store at room
temperature.
11. Ammonium sulfate solution: 12.5 % ammonium sulfate in
2.5 % phosphoric acid. Weigh 62.5 g of ammonium sulfate,
dissolve in around 400 mL of water, add 15 mL of 85 % phos-
phoric acid, and make up to 500 mL with water [
9
]. Store at
room temperature.
12. CBB staining solution: 0.08 % CBB G-250, 19 % ethanol, 10 %
ammonium sulfate, 2 % phosphoric acid. Mix 20 mL of CBB
G-250 solution and 80 mL of ammonium sulfate solution [
9
].
Mix immediately before use.
′
,
N
′
