Biomedical Engineering Reference
In-Depth Information
Fix the gel thoroughly either in TCA or 5-sulfosalicylic acid
(cf. Protocol 2.1.1) after electrophoresis to remove urea prior to
staining.
References
Swank RT, Munkres KD (1971) Anal Biochem 39:462
2.1.5 TRICINE-SDS-Polyacrylamide Gel Electrophoresis
for Proteins and Oligopeptides in the Range
of 1000-50 000 Daltons
The Schägger and von Jagow system is also suitable for small
proteins and polypeptides, especially for peptides resulting from
mapping experiments after bromocyan or tryptic cleavages. Be-
cause of its excellent separation power in the lower molecular
weight range, it is a good completion to the Swank and Munkres
protocol (Protocol 2.1.4). Which of these systems is used depends
on the analytical aim.
48% acrylamide (w/v), 1.5% N,N
-methylene bisacrylamide
A
Solutions/Reagents
(w/v)
A
46.5% acrylamide (w/v), 3.0% N,N
-methylene bisacrylamide
(w/v)
B3M Tris, adjusted with HCl to pH 8.45, 0.3% SDS (w/v)
C 10% TEMED (v/v) in ddH
2
O
D 10% ammonium persulfate (w/v)
E
anode buffer
:0.2M Tris-HCl, pH 8.9
F
cathode buffer
:0,1M Tr i s , 0 , 1 M Tricine (M
r
179.2), pH 8.25,
0.1% SDS (w/v)
G
sample buffer fourfold
: 150 mM Tris, 12% SDS (w/w), 50 mM
DTE or DTT, 30% glycerol (v/v), 0.05% (w/v) Coomassie Bril-
liant Blue G250, pH 7.0
Prepare the separation gel and the stacking gel according to Ta-
ble 2.6. The separation gel may be poured separately and overlaid
with ddH
2
O, but the stacking gel also can be carefully poured di-
rectly onto the fresh (liquid) separation gel.
For gels with %T
>
10 and %C
>
3 it is recommended to use
a separation gel between stacking and separation gel (about 1
/
5to
/
1
10 of the total gel length).
Dissolve the samples in buffer G (0.5 - 2
µ
µ
g
peptide per expected band, depending on the detection method).
Mix liquid samples in a ratio of 3 vol. of sample to 1 vol.ofbuffer
(fourfold), dissolve solid samples in 1:4 diluted buffer G, heat to
80
◦
C
9
for 10 min, and add the samples to form a layer below the
cathode buffer in the sample pockets of the gel.
g protein or 2 - 5
9
If urea is added to the sample buffer, heat only to 40
◦
C for 20 min.
