Biomedical Engineering Reference
In-Depth Information
in each case three-dimensional structures formed by the macro-
molecule's constituents) reconstitute during washing and blocking
of the blotting membrane, but sometimes the lack of an antibody
reaction is not due to the missing target macromolecule: the re-
quired epitopes are not presented under the distinct conditions.
Immunochemical detection is possible in one step, if the detect-
ing antibody carries the signal-forming principle. But also cascades
of steps are used to identify the first-bound antibody and by it the
antigen immobilized by blotting. Antibodies form these cascades,
i.e., if the first bound antibody is from species A, e.g., rabbit, a sec-
ond antibody from other species, e.g., goat, directed against the
primary, is used.
A second possibility is the modification (conjugation) of an
Biotin/
(strept)avidin
antibody by a label, e.g., biotin, which is detected later on by a spe-
cific receptor, e.g., (strept)avidin. In each case the last part of such
a cascade has to carry a measurable label. Such labels are enzymes,
fluorescent dyes, colloids, radioactive isotopes, paramagnetic sub-
stances, and others.
As indicator enzymes horseradish peroxidase (HRP or HRPO),
alkaline phosphatase (AP), or
β
-galactosidase, are favored, since
they are relatively robust, have a high product-forming rate, are
easy to purify, and are cheap. The most used colloids are from gold,
silver, and iron, and iodine isotopes are mostly taken as radioactive
labels in immunoassays.
The first step in immunochemical detection of proteins after
electrotransfer is blocking the support with an inert material to
inactivate further non-specific binding of protein. The blocking
reagent should cover the membranes at those areas where no blotted
protein is bound and should not react with any of the reactants of
immunochemical detection cascade as indicated by no non-specific
staining, i.e., resulting in blank background of the membrane.
The following protocol is an example for blocking, working very
well in numerous cases, but optimization by use of other blocking
reagents is worth checking every time.
0.05% Tween 20 (w/v), 0.1% serum albumin
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A
(w/v) in PBS
Solutions/Reagents
B
0.05% Tween 20 (w/v) in PBS
After electrotransfer the wet membrane is rocked in about 1 ml of
Soln. A per square centimeter of membrane at RT three times for
10 min each
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.
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Mostly bovine serum albumin (BSA) is used. Gelatin from cold
fish, inactivated calf serum, casein hydrolysate, non-fat dry milk,
polyvinylpyrrolidone, or Tween 20 are also suitable. Concentrations
from 0.1 to 0.5% (w/v) are sufficient. Casein and milk are not of first
choice if (strept)avidin conjugates are used.
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If anti-peptide antibodies are used the detection sensitivity is often in-
creased if the humid membrane is treated in a steam autoclave at 120
◦
C
for 30 min before blocking (Swerdlow PS, Finlay D, Varshavsky A
