Essential Cell Biology 5th edition

How Proteins Work
141
USING ANTIBODIES TO PURIFY MOLECULES
A
O
C
A
J S
mixture of molecules
M
L
IMMUNOPRECIPITATION
B K
E
Q
P
G
F N
A
H
D R
IMMUNOAFFINITY
COLUMN
CHROMATOGRAPHY
bead coated with
anti-A antibodies
mixture of molecules
R
B
C
A
E
Q
G
P
O
A
H
K
M
F
D
J S
L
N
A
A
elute antigen A
from beads
A
A
A
A
A
add specific
anti-A antibodies
A
collect aggregate of A molecules and
anti-A antibodies by centrifugation
column packed
with these beads
K
C
R
etc
N
discard flow-through
A
A
collect pure antigen A
MONOCLONAL ANTIBODIES
Large quantities of a single type of antibody
molecule can be obtained by fusing a B cell
(taken from an animal injected with antigen A)
with a tumor cell. The resulting hybrid cell
divides indefinitely and secretes anti-A
antibodies of a single (monoclonal) type.
B cell from animal
injected with antigen
A makes anti-A
antibody but does
not divide forever.
Tumor cells in
culture divide
indefinitely but
do not make
antibody.
FUSE ANTIBODY-SECRETING
B CELL WITH TUMOR CELL
Hybrid cell
makes and
secretes anti-A
antibody and
divides
indefinitely.
USING ANTIBODIES AS MOLECULAR TAGS
MICROSCOPIC DETECTION
BIOCHEMICAL DETECTION
specific antibodies
against antigen A
couple to fluorescent dye,
gold particle, or other
special tag
Antigen A is
separated from
other molecules
by electrophoresis.
Note: In all cases, the sensitivity can
be greatly increased by using multiple
layers of antibodies. This “sandwich”
method enables smaller numbers of
antigen molecules to be detected.
labeled antibodies
50 µm 200 nm
Fluorescent antibody binds to Gold-labeled antibody binds to
antigen A in tissue and is detected antigen A in tissue and is detected
in a fluorescence microscope. The in an electron microscope. The
antigen here is pectin in the cell antigen is pectin in the cell wall
walls of a slice of plant tissue. of a single plant cell.
Incubation with the
labeled antibodies
that bind to antigen A
allows the position of the
antigen to be determined.
Labeled second antibody
(blue) binds to first
antibody (black).
antigen
cell
wall
ECB5 Panel 4.03b/panel 4.03b