Biomedical Engineering Reference
In-Depth Information
B .2M sodium phosphate buffer, pH 7.2, 0.2% SDS (w/v) (
do
not use potassium phosphate
) (7.8 g NaH
2
PO
4
·
H
2
O, 38.6 g
Na
2
HPO
4
·
7H
2
Oand2g SDS in 1000 ml ddH
2
O)
C
TEMED (v/v) in ddH
2
O (stable at RT)
D
10% ammonium persulfate (w/v) in ddH
2
O (stable for 2 -
3daysat4
◦
C)
E
sample buffer
:10mM sodium phosphate buffer, pH 7.2, 2%
SDS (w/v), 4% 2-mercaptoethanol (v/v)
For preparing the separation gel, pipette the solutions in order
given in Table 2.4, adjust the required volume with ddH
2
O, and
mix well (avoid foam forming). Start the polymerization reaction
by addition of Soln. D. Introduce the mixture into the cassette
without air bubbles up to about 10 mm from the top of the front
plate and gently layer ddH
2
O or n-butanol to keep a smooth surface.
Polymerization needs 20 - 30 min and may be prolonged by cooling
or decreasing the amount of Soln. D, and vice versa.
Just before performing the electrophoresis, carefully remove
the liquid above the gel with filter paper and prepare the stacking
gel. Since it is a continuous separation system, the stacking gel may
be poured immediately after introducing the separation gel instead
of covering with ddH
2
O or n-butanol, but getting sharp separations
between both gels is a somewhat sophisticated process.
Prepare the sample in buffer E with a protein concentration not
more than 20 mg
ml. Heat the solution to 95
◦
C for 2 - 3 min and
supplement with some crystals of sucrose or a droplet of glycerol
or 1/10 volume of bromophenol blue in 50% sucrose solution.
Mix the dissolved sample with an equal volume of double con-
centrated sample buffer E.
If the sample has an (expected) ionic strength
>
0.05 it has to
dialyze against sample buffer because this system is exceptionally
sensitive to ions.
The minimal amount of loaded protein depends on the detec-
tion method. As a rule of thumb, for Coomassie staining at least
1
/
µ
g of protein per band is needed.
Table 2.4.
Casting protocol for the W
EBER
,P
RINGLE
, and O
SBORN
system
(C =
2.65
%)
%T = 20
15
10
7.5
5
Stacking gel
Solution
(ml/10 ml)
A
4.40
3.30
2.20
1.64
1.10
0.72
B
5.00
5.00
5.00
5.00
5.00
5.00
C
0.10
0.10
0.10
0.10
0.10
0.10
Add H
2
Oto9.9ml
D
0.10
0.10
0.10
0.10
0.10
0.10
