Protein Protocols Protein Protocols

Blotting–Electroblotting 317
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Protein Blotting by Electroblotting
Mark Page and Robin Thorpe
1. Introduction
Identification of proteins separated by gel electrophoresis or isoelectric-focusing is
often compounded by the small pore size of the gel, which limits penetration by macromolecular
probes. Overcoming this problem can be achieved by blotting the proteins
onto an adsorbent porous membrane (usually nitrocellulose or diazotized paper), which
gives a mirror image of the gel (1). A variety of reagents can be incubated with the
membrane specifically to detect and analyze the protein of interest. Antibodies are
widely used as detecting reagents, and the procedure is sometimes called Western blotting.
However, protein blotting or immunoblotting is the most descriptive.
Electroblotting is usually preferred for immunoblotting in which proteins are transferred
to the membrane support by electrophoresis (2). A possible exception to this is in
transfer from isoelectric focusing gels, where the proteins are at their isoelectric points
and uncharged. Therefore, there is considerable delay before the proteins start to
migrate in an electric field; also they can migrate in different directions and their rate
of transfer can vary. For these reasons, transfer from isoelectric focusing gels by capillary
blotting (see Chapter 41) may be preferable, particularly if very thin gels are used.
Electroblotting has the advantage that transfer takes only 1–4 h, and lateral diffusion
of proteins (which causes diffuse bands) is reduced. Overall, nitrocellulose membranes
are recommended. These are efficient protein binders and do not require activation, but
are fragile and need careful handling. Nitrocellulose membranes, such as Hybond-C
extra, are more robust. If the antigens of interest do not bind efficiently or if the blot is
to be reused, then diazotized paper, or possibly nylon membranes, can be used. A suitable
electrophoretic transfer chamber and power pack are required; these are available
commercially or can be made in a laboratory work shop. The apparatus consists of a
tank containing buffer, in which is located a cassette, clamping the gel and membrane
tightly together. A current is applied from electrodes situated at either side of the cassette.
The buffer is often cooled during transfer to avoid heating effects.
2. Materials
1. Transfer apparatus.
2. Orbital shaker.
3. Nitrocellulose sheet, 0.45-µm pore size, e.g., Hybond-C extra.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
317