Electrophoresis - Basic Methods for the Biochemical Lab - page 30

Important! Cool the whole polyacrylamide gel during electrophore-
sis, since most of the electric power produces

Coulomb heat result-

ing in “smiling” bands.

The electrophoresis is finished when the tracking dye has reached
about 5 mm above the bottom of the gel. Turn off the power supply,
open the cassette carefully, and submerse the gel into a tenfold of gel
volume of fixation solution [15% trichloroacetic acid (w/v), 15% 5-

Protein fixation

sulfosalicylic acid (w/v), or glacial acetic acid/methanol/H 2 O (1:3:6,
v/v/v)] or mount the gel into the electrotransfer apparatus.
If necessary, store the gel within the cassette or wrapped with
plastic film in a refrigerator overnight, but avoid freezing.

References

Laemmli UK (1970) Nature 227:680

2.1.2 SDS-Polyacrylamide Gel Electrophoresis
at Neutral pH (NuPAGE)

Some proteins with posttranslational modifications are sensitive
even with respect to the slight alkaline pH of the Laemmli system.
For example, phosphate groups of phosphoproteins may hydrolyze
or SH groups are possibly oxidized. To prevent this, the neutral pH
SDS-PAGE system is used. Further advantages are shorter separa-
tion time by using higher voltages and a higher separation power.
NuPAGE 8 gels and buffers are available ready to use, but they are
also easily self-made.

39% acrylamide (w/v), 1% N,N -methylene bisacrylamide

A

Solutions/Reagents

(w/v)
B . 6M Bis-Tris (M r 209.24), 1.71 M HCl (3.84 ml 37% HCl per
25 ml), adjusted to pH 6.5
C 10% TEMED (v/v) in ddH 2 O(stabileatRT)
D 10% ammonium persulfate (w/v) in ddH 2 O (stabilize for
2-3 days at 4 ◦ C)
E MES electrophoresis buffer 20-fold :1M MES (M r 195.2), 1 M
Tris, 2% SDS (w/v), 0.6% EDTA (free acid) (w/v), pH 7.3
F MOPS electrophoresis buffer 20-fold :1M MOPS (M r 209.3),
1 M Tris, 2% SDS (w/v), 0.6% EDTA (free acid) (w/v), pH 7.7
G sample buffer fourfold : 0.986 M Tris, 40% sucrose (w/v), 8%
SDS (w/v), 0.06% EDTA (w/v), 0.075% (w/v) Coomassie Bril-
liant Blue R250, 0.25%) phenol red (w/v)
H blottingbuffer20-fold :0.5MBicine (N,N-bis(2-hydroxyethyl)-
glycine), 0.5 M Bis-Tris, 20 mM EDTA
Mix the gel according to Table 2.3 and pour it into the cassette.
Since a stacking gel is not necessary, put the comb directly into

8

Invitrogen Corp./NOVEX