Biomedical Engineering Reference
In-Depth Information
Fig. 4.3.
Patternof counterelectrophoresis.
Ag
antigenwell,
Ab
antibody-
containing well. + Anode
/
cm for about 45 min.Itis
recommended to cool the slide during electrophoresis.
When electrophoresis is finished, cover the gel with dry filter
paper and several layers of tissue paper and press by a weight.
Remove the paper after 15 min and agitate the slide in PBS for 2
Run electrophoresis with 20 - 40 V
×
30 min. Stain the gel as described in Protocol 4.8.4. The precipitin
bands appear as fine blue lines.
References
Hudson L, Hay FC (1989) Practical immunology, 3rd ed. Blackwell, London,
p 241
4.12 Dot-Blot Assay
Mostly plastics, such as nitrocellulose, polyvinylidene fluoride
(PVDF), or polystyrene (used for microtiter plates), are used for
immobilization of antigens. Because these materials interact with
proteins by different mechanisms, sometimes it occurs that the
binding material induces or removes epitopes necessary for bind-
ing of specific antibodies. So, if no reaction between antigen and
antibody is observed, repeat the experiment by using another
support (e.g., Nunc offers several types of polystyrene microtiter
plates with different binding properties) or bind the antigen via
a spacer (e.g., by biotinylation of antigen and immobilization of
streptavidin on the support). A dubious positive result should be
Test of specifity by
competition
checked, if possible, by a competition experiment of incubation of
the antibody-containing solution in the presence of dissolved (free)
antigen.
A
0.1% gelatin (w/v) or 5% heat-inactivated calf serum or 0.1%
Solutions/Reagents
serum albumin (w/v) or 0.2% non-fat milk powder (w/v) or
0.1% Tween 20 (w/v) in PBS
B
secondary antibody-HRP conjugate dilution in PBS (dilution
for instance 1:1000 to 1:50 000)
