Biomedical Engineering Reference
In-Depth Information
18.25 g acrylamide, 0.487 g N,N
-methylene bisacrylamide in
A
Solutions/Reagents
50 ml ddH
2
O
B . 5M Tr i s
·
HCl, pH 8.9
C
HCl, pH 6.8
D . 5M Tr i s , 0 . 3 8 M glycine, adjusted with HCl to pH 8.3
E 10% TEMED (v/v) in ddH
2
O
F 10% ammonium persulfate (w/v) in ddH
2
O
Solutions are gently mixed according to Table 2.9 in the indicated
order and filled up with ddH
2
O. Start the polymerization by ad-
dition of Soln. F and pour the mixture immediately into the gel
cassette. When the appropriate height is reached, cover the liquid
with water or n-butanol to get a smooth surface. Prepare the stack-
ing gel as short as possible before starting the electrophoresis to
avoid a decrease of the pH jump between stacking and separation
gel by diffusion.
ElectrodebufferisSoln.D,thesamplebufferispreparedby
addition of a trace bromophenol blue and sucrose to Soln. D. Elec-
trophoretic conditions are similar to that given in Protocol 2.1.1.
0.125 M Tr i s
·
References
Davis BJ (1964) Ann NY Acad Sci 121:404
Table 2.9.
Casting protocol for native PAGE
Stacking gel
a
Separation gel
b
Solution
(ml/10 ml)
A
1.2
2.0
B
-
5.0
C
5.0
-
E
0.1 0.1
Add H
2
Oto9.9ml
F
0.1
0.1
a
%T=4.5,%C=2.6
b
%T = 7.5, %C = 2.6
2.1.9 Cathodic Discontinuous Polyacrylamide Gel
Electrophoresis (Native PAGE)
This protocol is adapted for analysis of proteins with pI
>
8. The
other deviations from SDS-PAGE are described in Protocol 2.1.8.
Attention!
Migration is from “
+
”to“
−
”, i.e., the opposite to SDS-
PAGE or anodic systems.
Basic fuchsin (rosaniline, basic violet 14) or Pyronin Y are suitable
as tracking dye.
