Biology Reference
In-Depth Information
IEF
MPMT
IEF
MPMT
IEF
MPM T
IEF
MPM T
TMB
DAB
a
-Chloro-naphthol
Guaiacol
Fig.
3
In-gel staining of peroxidases after separation of membranes by native PAGE. Peroxidase activities were
visualized by “specifi c” stains after separation of microsomal proteins (50
μ
g) by native IEF-PAGE (pH 3-10)
or after separation of washed membrane fractions (50
g protein) by hrCNE. TMB detect
heme
- and copper-
containing proteins, whereas DAB and the phenolic substrates
μ
-chloro-naphthol and guaiacol react with class
III peroxidases. Time of development was 2-3 min for all stains. A shorter time of incubation with substrates
may increase the resolution in the upper part of the TMB or guaiacol lanes and reveal additional protein bands
in this range, but in that case protein bands with lower abundance or activity will disappear.
M
microsomal
fraction,
PM
plasma membrane,
T
tonoplast
α
Fig.
4
2D-PAGE (hrCNE/modSDS-PAGE) of membrane fractions. Proteins (250
g) of washed microsomal frac-
tions (
a
,
c
) and plasma membranes (
b
,
d
) were separated by hrCNE in the fi rst dimension and by modSDS-
PAGE in the second dimension. Guaiacol (
a
,
b
) or TMB (
c
,
d
) were used to visualize peroxidases,
heme
and
copper containing proteins. For an easier identifi cation of spots on the gel images may be inverted
μ
