Biomedical Engineering Reference
In-Depth Information
B 14.5% acetic acid (w/v), 5.4% TEMED (v/v) in ddH
2
O
C4M urea in ddH
2
O
D
electrode buffer
:2.5M urea, 0.9 M acetic acid
Acrylamide and urea must be ultra-pure. Their aqueous solu-
tions are not allowed to warm above 35
◦
C.
The gel resulting from Table 2.8 has %T
=
=
15.1 and %C
0.66.
A stacking gel is not required.
Prior to protein separation, a pre-electrophoresis is essential.
It is done overnight with 3.5 mA
/
cm
2
(constant current). The elec-
trode buffer D is renewed after pre-electrophoresis.
The samples are dissolved in buffers C or D, supplemented with
some crystals of sucrose.
The electrophoresis is run at 8 - 10 V
/
cm (cv), the proteins mi-
grate from “−” to “+”. The electrophoresis front may be indicated
by neutral red.
References
Panyim S, Chalkley R (1969) Biochem Biophys Res Comm 37:1042
Table 2.8.
Casting protocol for the acidic urea PAGE
Solution
(ml/10 ml)
A 2.50
B 1.25
C 6.25
Ammonium persulfate
(added as solid)
12.5 mg
2.1.8 Anodic Discontinuous Polyacrylamide Gel
Electrophoresis (Native PAGE)
If proteins with acidic or nearly neutral pI shall be electrophoreti-
cally analyzed without destroying (denaturating) biological activ-
ity, this PAGE system may be used. In contrast to SDS-containing
systems, which sieve according to molecular size, the separation
principle is electrophoretic mobility, i.e., molecular size (“molar
mass”) is not the primary quantity; netto charge at the given pH is
more dominant.
Quaternary structures or ligand-ligate (receptor) interactions
may be partially conserved during electrophoresis. Identification
of a distinct protein is possible only by biochemical or immuno-
chemical reactions or by comparison with an authentic sample.
Most proteins migrate from cathode (“−”) to anode (“+”). As
tracking dye for indicating the electrophoresis front may serve
bromophenol blue.
