Biomedical Engineering Reference
In-Depth Information
Table 2.6.
Casting protocol
for TRICINE-SDS-PAGE.
(According to
S
CHÄGGER
and
VON
J
AGOW
)
Separation gel
Stacking
Spacer
T% = 10
%T = 16.6
%T = 16.5
gel
a
%C=6
b
gel
C%=3
%C=3
Solution
(ml)
A
0.80
2.00
2.00
3.33
-
A
-
-
-
-
3.33
2.5
b
B
3.33
3.33
3.33
3.33
C
0.075
0.05
0.05
0.033
0.033
Glycerol
-
-
1.0
1.0
1.0
Add H
2
Oto10ml
D
0.10
0.05
0.05
0.033
0.033
(V/cm gel length)
cv during run
8 - 11
6 - 7
6.5 - 7.5
a
Addition of a trace of Coomassie Brilliant Blue R250 facilitates the
sample application and indicates the electrophoresis front
b
For peptides
<
5 kD
As tracking dye Coomassie Brilliant Blue G-250 is recom-
mended. Bromophenol blue does not suit well since it moves sig-
nificantly slower than small peptides.
Run the electrophoresis at room temperature as indicated in
Ta b l e 2 . 6 .
After the run common fixation and staining protocols are used.
For Western blotting according to the Kyhse-Anderson semi-
dry protocol (Protocol 2.4.3), the following buffers are recom-
mended by Schägger and v.
J a g ow :
ε
-aminohexanoic acid, 30 mMTr i s , p H 8 . 6 ;
anode buffer
:0.3M Tr i s , 0 . 1 M TRICIN, pH 8.7; it is possible to
supplement with 20% methanol (v/v).
cathode buffer
:0.3M
References
Schägger H, v. Jagow G (1987) Anal Biochem 166:368
Jagow G von, Schägger H (1994) A practical guide to membrane protein
purification. Academic Press, San Diego, p 65
2.1.6 SDS-Polyacrylamide Gel Electrophoresis at pH 2.4
This system allows the separation of alkali-labile proteins (e.g.,
acylphosphate phosphoproteins) under denaturating conditions
according to their molar mass. Despite the low acrylamide concen-
tration (%T
=
=
3.61), the separation force is remarkable.
Because it is a SDS-containing system, the migration is from “−” to
“+” despite the low pH.
5.61, %C
