Biomedical Engineering Reference
In-Depth Information
molar mass, and a normal movement in electrophoresis, e.g., linear
correlation with respect to other standard proteins. Not all proteins
given in Table 2.12 fulfill these criteria; therefore, an additional table
(Table 2.13) is given with proteins often used in SDS-PAGE.
Since the obtained optical density (intensity of stain) per mass
unit of a protein differs depending on its nature and the staining
method, for some commercial available protein mixtures the com-
position is given, which produces staining a homogeneous picture
by Coomassie.
About 1 - 5
µ
l of the pre-mixed ready-to-use protein standards
per lane are recommended in SDS-PAGE combined with Coomassie
staining.
2.3.3 Covalently Colored Marker Proteins
Protein molar mass standards covalently labeled with dyes are valu-
able when the electrophoresis is followed by a Western blot or when
electrophoresis has to be monitored. Furthermore, procedures of
gel staining alter the geometry, and assignment of bands on blot to
marker bands within the gel is sometimes difficult.
The electrophoretic mobility of the labeled proteins obtained by
this protocol is practically not different from that of the unlabeled
proteins.
A .1M borate buffer, 5% SDS (w/v), pH 9.0
Solutions/Reagents
B 0mM dimethylamino-azobenzen-4
-sulfonyl chloride (dab-
syl chloride, M
r
323.8) in acetone
C
electrophoresis sample buffer
:50mM Tris-HCl, 4% SDS (w/v),
5% 2-mercaptoethanol (v/v), 10% sucrose or glycerol (w/v),
pH 6.8.
The respective marker proteins, single or mixed, are dissolved in
Soln. A to a concentration of 20 mg
/
ml at RT. Then 0.5 - 1 vol.of
Soln. B are added and the mixture is heated to 60
◦
C for 5 min.
After cooling, the labeled proteins are lyophilized without fur-
ther purification and dissolved again in Soln. C to a concentration
of 20 mg
/
ml. If the assay was larger than 0.1 ml, the product is
aliquoted and stored at −20
◦
C.
The remaining dabsyl chloride is not removed, since it reacts
with Tris of Soln. C to a dye acting as tracking dye like bromophenol
blue.
1to2
µ
g per lane of the obtained labeled marker proteins each
are applied.
Several manufacturers distribute ready-to-use colored marker
proteins (cf. example in Table 2.13).
References
Tzeng M-C (1983) Anal Biochem 128:412
Lin J-K, Chang J-Y (1975) Anal Chem 47:1634
