Biology Reference
In-Depth Information
6. Fill both chambers with running gel buffer. Electrophoresis is
carried out at 30 mA per gel up to 200 V at 4 °C until the blue
front is running down.
7. When the electrophoresis is fi nished the gel is transferred into
the according buffer (Subheading
3.9
).
3.9 Differential
In-Gel Staining for
Peroxidases
The equilibration steps and stainings are accomplished on a hori-
zontal shaker. All in-gel stainings are scanned before the bands
become saturated (Figs.
3
and
4
).
1. The gel is transferred into 50 mL TMB-staining solution and
the box is covered in aluminum foil (TMB is light sensitive).
Keep the gel at a horizontal shaker for ~60 min.
2. Reaction is started by the addition of 0.1 % H
2
O
2
(180
3.9.1 TMB-Staining
L).
After few seconds turquoise TMB bands appear. When the
desired band intensity is reached, the reaction is stopped by the
buffer exchanged against 30 % 2-propanol/70 % 250 mM
sodium acetate buffer, pH 5.0.
μ
1. The gel is equilibrated for 10 min in 50 mL 50 mM Na-acetate,
10 mM CaCl
2
, pH 5.0.
2. 80 mg DAB are solved in 1 mL DMSO and added to 49 mL
50 mM Na-acetate, 10 mM CaCl
2
, pH 5.0. The staining solu-
tion is mixed.
3. The equilibration solution is exchanged against the staining
solution and the gel is equilibrated for 10 min in the solution.
4. The reaction is started by the addition of 0.5 % H
2
O
2
(900
3.9.2 DAB-Staining
L).
Brown bands appear after a few minutes. When the desired
band intensity is reached, the gel is scanned.
μ
1. The gel is equilibrated in 50 mL 0.1 M Na
2
HPO
4
buffer,
pH 6.5 for 15 min.
2. The buffer is exchanged against
3.9.3 α-Chloro-
Naphthol-Staining
-chloro-naphthol-staining
solution and again equilibrated for 10 min.
3. The reaction is started with 0.1 % H
2
O
2
(180
α
L). Violet bands
appear after a few minutes. When the desired band intensity is
reached, the gel is scanned.
μ
1. The gel is transferred into 50 mL 50 mM Na-acetate, pH 5.0
with 10 mM CaCl
2
and equilibrated for 10 min.
2. 0.5 % Guaiacol (250
3.9.4
Guaiacol-Staining
μ
L) is added to the buffer. Equilibrate
again 10 min.
3. 0.15 % H
2
O
2
(250
L) is added to the staining solution. After
few seconds the fi rst orange bands become visible. When the
desired band intensity is reached, the gel is scanned.
μ
