Biomedical Engineering Reference
In-Depth Information
Table 2.3.
Casting protocol for neutral-pH SDS-PAGE (%C = 2.5)
%T = 4
8
10
12
Solution
(ml/10 ml)
A
1.00
2.00
2.50
3.00
B
1.25
1.25
1.25
1.25
C
0.02
0.02
0.02
0.02
Add H
2
O to 9.98 ml
D
0.02
0.02
0.02
0.02
the separation gel. After complete polymerization, the gel is ready
for use.
Mount the gel into the electrophoresis apparatus; fill the elec-
trode chambers with 1:20 diluted MES or MOPS electrode buffer.
A concentration gradient gel with 4
12%T running with MES
buffer separates between 2 and 250 kD; performing with MOPS
buffer the separation range is between 10 and 250 kD.
Mix samples with one-third of their volume of buffer G and
heat at about 70
◦
C for 5 min. For cleavage of disulfide bridges add
DTTtoafinalconcentrationof10mM.
Runagelof8cm separation length with 200 V cv for 50 -
60 min. Surround the gel completely by electrode buffer during
electrophoresis to carry off the heat.
After electrophoresis, the gel is fixed and stained as other PAGE
gels, too. For semi-dry blotting the 1:20 diluted buffer H is recom-
mended.
→
References
NuPAGE Electrophoresis System. Instruction Booklet (1997) NOVEX, San
Diego, Calif.
2.1.3 SDS-Polyacrylamide Gel Electrophoresis According
to W
EBER
,P
RINGLE
,andO
SBORN
This PAGE is a continuous system with respect to pH. Its reso-
lution is lower than that of a disc system. The advantage lies in
the use of buffers free of primary amino groups; therefore, it is
recommended if an electrotransfer is intended onto chemical reac-
tive supports because a buffer change decreases transfer yield and
separation performance (broadening of bands by diffusion during
buffer change).
44.4% acrylamide (w/v), 1.2% N,N
-methylene bisacrylamide
A
Solutions/Reagents
=
(w/v) in ddH
2
O(%C
2.65)
