Biomedical Engineering Reference
In-Depth Information
Wash the blot after the antibody and conjugation incubation steps
with Soln. A, followed by an equilibration in 2 ml
/
cm
2
of Soln. B
for 2 - 3 min.
Dilute Soln. C 1:100 with Soln. B.
Substitute Soln. B by the AMPPD dilution (0.2 ml
/
cm
2
)and
incubate for 5 min. Drain the blot and wrap it with a solvent-tight
foil. Incubate the wrapped membrane at 37
◦
C for 5 min,andthen
expose the blot face towards to the film in an X-ray film cassette for
15 min to 24 h or photograph it with a digital camera.
It is possible to make a first exposure for, for example, 15 min,
followed by a second and/or third with longer exposing times on
new films each.
2.5.6 Carbohydrate-Specific Glycoprotein Detection
After Electrotransfer
Specific detection of oligosaccharides is possible using lectins,
carbohydrate-binding proteins from very different sources. A se-
Table 2.16.
Selected lectins
Lectin
a
Abbre-
MW
Specific carbohydrate
(kD)
b
viation
α
1-6)GlcNac
c
Aleuria aurantia
lectin
AAA
Fuc(
α
α
Concanavalin A
Con A
102
-D-Man,
-D-Glc
β
Datura stramonium
lectin
DSA
86
Gal(
1-4)GlcNAc
α
Galanthus nivalis
lectin
GNA
52
Man(
1-3)Man
α
Gorse lectin
UEA I
68
-Fuc
Gorse lectin
UEA II
68
(GlcNAc)
2
α
α
Lentil lectin
LcL
49
-D-Man,
-D-Glc
α
Maackia amurensis
lectin
MAA
130
NeuNAc(
2-3)Gal
β
Mistletoe lectin
VaL
115
-D-Gal
β
Peanut lectin
PNA
120
-D-Gal
1−3
-D-GalNAc
Phytohemagglutinin
PHA-E
128
(non-specific)
Phytohemagglutinin
PHA-L
126
(non-specific)
β
Phytolacca americana
lectin
PWM
32
GlcNAc(
1-4)GlcNAc
1−5
β
Ricinus lectin I
RCA
120
120
-D-Gal
Ricinus lectin II
RCA
60
60
D-GalNAc
α
Sambucus nigra
lectin
SNA
140
NeuNAc(
2-6)Gal
α
Snail lectin
HPA
79
-GlcNAc
α
Soybean lectin
SBA
110
-GalNAc
Wheat germ agglutinin
WGA
36
[D-GlcNAc]
n
,NeuNAc
a
The terms “lectin” and “agglutinin” are used synonymously
b
Molar mass under physiologic conditions
c
Fuc
fucose,
Gal
galactose,
GalNAc
N-acetylgalactosamine,
Glc
glucose,
Glc-
NAc
N-acetylglucosamine,
Man
mannose,
NeuNAc
N-acetylneuraminic
acid (sialic acid, NANA)
