Page 77 - Academic Press Encyclopedia of Physical Science and Technology 3rd Molecular Biology

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Encyclopedia of Physical Science and Technology EN007I-331 July 3, 2001 18:42

688 Immunology—Autoimmunity

FIGURE 3 Diagrammatic representation of apparatus used in electrophoretic transfer of proteins from polyacrylamide
gel to nitrocellulose. The transfer assembly consists of a “sandwich” made up of an outer porous fiber pad overlaid
with several sheets of adsorbent paper, the polyacrylamide gel, a sheet of nitrocellulose, and then an additional layer
of adsorbent paper sheets and another porous fiber pad. Assembly is done with all the components saturated with
buffer to avoid air bubbles that may hinder protein transfer, particularly when between the acrylamide gel and the
nitrocellulose sheet. The sandwich is held together by clamps or other firm support and immersed in a buffer-filled
tank. Applying voltage results in the proteins being electrophoretically transferred from the polyacrylamide gel to the
nitrocellulose. In the case of SDS-PAGE, the negatively charged proteins migrate toward the anode.

exact mechanism that allows proteins to bind to nitrocel- sence of significant SDS in the transfer buffer, as SDS is
lulose is largely unknown, although hydrophobic forces known to reduce protein adsorption to nitrocellulose.
may contribute. Under conditions of sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), 3. Method
proteins are denatured with SDS and become negatively
charged. The passing of an electric current through the An immunoblotting protocol consists of numerous steps,
polyacrylamide gel forces proteins to migrate toward the from SDS-PAGE resolution of a mixture of antigens to
positive anode, with the proteins being resolved accord- interpretation of the results (Fig. 4). For simplicity we
ing to their molecular weight by virtue of the porosity of consider here only those steps relating to SDS-PAGE res-
the gel. Transfer of the resolved proteins to nitrocellulose olution, electrophoretic transfer, and immunological de-
occurs according to essentially the same principle. Under tection of autoantigens. The source of protein for SDS-
an electric current the negatively charged proteins migrate PAGE analysis should be one that contains the antigen or
out of the polycarylamide gel and onto the nitrocellulose antigens of interest. One means of achieving this is to use a
(Fig. 3). An important feature of the technique is the ab- cellular substrate that has been found suitable for detection