Biology Reference
In-Depth Information
2.8 Modifi ed
SDS-PAGE (modSDS-
PAGE) Components
1. 4× Resolving gel buffer: 1.5 M Tris-HCl with 0.4 % SDS,
pH 8.8. Weigh 90.86 g Tris and transfer it to a 500 mL volu-
metric fl ask. Add about 350 mL water to the fl ask and solve the
Tris. Weigh 2 g SDS and add it carefully to the Tris solution.
When the SDS is solved adjust pH with HCl (
see
Note 3
).
Make up to 500 mL with water.
2. 4× Stacking gel buffer: 0.5 M Tris-HCl with 0.4 % SDS,
pH 6.8. Weigh 30.2 g Tris and prepare a 500 mL solution as
in previous step.
3. 10× SDS-PAGE electrophoresis buffer: 0.25 M Tris, 1.92 M
glycine, 1 % SDS.
4. Loading buffer (SDS-LB): 125 mM Tris-HCl, 0.2 % (w/v)
SDS, 20 % (w/v) glycerol, and 0.004 % (w/v) bromo-phenol,
pH 6.8.
5. Equilibration buffer for the second dimension: 1× stacking gel
buffer with 1 % SDS plus 10 % glycerol and a trace of
Bromophenol blue.
2.9 First Dimension
modSDS-PAGE
1. First dimension modSDS-PAGE: 4 and 18 % acrylamide resolv-
ing gel mixture (each 5 mL): Solution I: Mix 0.625 mL
AB-solution, 1.25 mL resolving gel buffer (4×), 0.303 g urea,
and 3.125 mL water. Solution II: Mix 2.92 mL AB-solution,
1.25 mL resolving gel buffer (4×), 0.303 g urea and fi ll up
with glycerol to 5 mL. Mix well and degas both solutions
before use.
2. 4 % Acrylamide stacking gel mixture for the fi rst dimension
(5 mL): Mix 0.625 mL AB-solution, 1.25 mL stacking gel
buffer (4×), 0.303 g urea and 3.125 mL water, degas it and
store cold.
1. Second dimension after hrCNE: 4 and 16 % acrylamide resolv-
ing gel mixture (each 5 mL): Solution I: Mix 0.625 mL
AB-solution, 1.25 mL resolving gel buffer (4×) and 3.125 mL
water. Solution II: Mix 2.67 mL AB-solution, 1.25 mL resolv-
ing gel buffer (4×) and fi ll up with glycerol to 5 mL. Degas
both solutions before use.
2. Second dimension after native IEF: 4 and 18 % acrylamide
resolving gel mixture (each 5 mL): Solution I: Mix 0.625 mL
AB-solution, 1.25 mL resolving gel buffer (4×) and 3.125 mL
water. Solution II: Mix 2.92 mL AB-solution, 1.25 mL
resolving gel buffer (4×) and fi ll up with glycerol to 5 mL.
Degas both solutions before use.
3. 4 % Acrylamide stacking gel mixture for the second dimension
(5 mL): Mix 0.625 mL AB-solution, 1.25 mL stacking gel
buffer (4×) and 3.125 mL water, degas it and store cold.
2.10 Second
Dimension
modSDS-PAGE
