Biomedical Engineering Reference
In-Depth Information
References
Allen RC, Saravis CA, Maurer HR (1984) Gel electrophoresis and isoelectric
focusing of proteins. Selected techniques. W. de Gruyter, Berlin
Chrambach A, Dunn MJ, Radola BJ (eds.) (1987 foll.) Advances in elec-
trophoresis, vol. 1 foll., VCH, Weinheim
Hames BD, Rickwood D (eds.) (1990) Gel electrophoresis of proteins: a prac-
tical approach, 2nd ed., Oxford University Press, New York
Deyl Z, Chrambach A, Everaerts FM, Prusik Z (eds.) (1983) Electrophoresis.
A survey of techniques and applications. J Chromatogr Library, vol.
18B, Elsevier, Amsterdam
Westermeier R (1997) Electrophoresis in practice: guide to methods and
applications of DNA and protein separations. 2nd ed., VCH, Weinheim
2.1.1 L
AEMMLI
SDS-Polyacrylamide Gel Electrophoresis
The Laemmli SDS-PAGE protocol is one of the most important an-
alytical techniques in analytical protein separation. It is a system
with discontinuous pH gradient (disc electrophoresis) and consists
of a stacking and a separation gel different in acrylamide concentra-
tion and pH. The separation gel may be formed with homogenous
acrylamide concentration or with an increasing gradient.
Molar mass determinations based on SDS-PAGE is sometimes
misleading, since some proteins are not conversed completely into
a rod-like shape or the protein/SDS ratio differs from the average.
40.0% acrylamide (w/v), 1.08% N,N
-methylene bisacrylamide
Solutions/Reagents
A
=
(w/v) in ddH
2
O; (%C
2.63)
A
40.0% acrylamide (w/v), 2.1% N,N
-methylene bisacrylamide
(w/v) in ddH
2
O; (%C
=
5.0)
B1M Tr i s
·
HCl, pH 8.8
HCl, pH 6.8
4
C .5M Tr i s
·
20% SDS (w/v) in ddH
2
O (store at RT, below 10
◦
C precipitation
occurs)
D
E
10% TEMED (v/v) in ddH
2
O
F
10% ammonium persulfate (APS) (w/v) in ddH
2
O (stabilize
for1dayat2-8
◦
C in a closed tube)
G
sample buffer (reducing buffer)
:50mM Tr i s
·
HCl, pH 6.8, 4%
SDS(w/v),10mM DTE
5
(v/v), 10 mM Na
2
-EDTA
4
Addition of 0.01% bromophenol blue to buffer C makes it easier to
recognize of the slots of the stacking gel and also allows the identification
of the electrophoresis front during run.
5
DTE/DTT (Cleland's ragent) as reducing reagent is recommended in-
stead of 2-mercaptoethanol, because the latter often forms pseudobands
especially in silver-stained gels. Forming of new disulfide bonds by
air or peroxidisulfate is suppressed by addition of 10 mM (final con-
centration) N-methylmaleiimide (NEM) or iodoacetamide. A further
selcective reduction reagent is tris(2-carboxethyl)-phosphonium chlo-
ride (TCEP
·
HCl), 100 mM stock solution in H
2
O, final concentration
