Protein Protocols Protein Protocols

Semidry Protein Blotting 321
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Protein Blotting by the Semidry Method
Patricia Gravel
1. Introduction
Protein blotting involves the transfer of proteins to an immobilizing membrane. The
most widely used blotting method is the electrophoretic transfer of resolved proteins
from a polyacrylamide gel to a nitrocellulose or polyvinylidene difluoride (PVDF) sheet
and is often referred to as “Western blotting.” Electrophoretic transfer uses the driving
force of an electric field to elute proteins from gels and to immobilize them on a matrix.
This method is fast, efficient, and maintains the high resolution of the protein pattern
(1). There are currently two main configurations of electroblotting apparatus: (1) tanks
of buffer with vertically placed wire (see Chapter 39) or plate electrodes and (2) semidry
transfer with flat-plate electrodes.
For semidry blotting, the gel and membrane are sandwiched horizontally between
two stacks of buffer-wetted filter papers that are in direct contact with two closely
spaced solid-plate electrodes. The name semidry refers to the limited amount of buffer
that is confined to the stacks of filter paper. Semidry blotting requires considerably less
buffer than the tank method, the transfer from single gels is simpler to set up, it allows
the use of multiple transfer buffers (i.e., different buffers in the cathode and anode
electrolyte stacks) and it is reserved for rapid transfers because the amount of buffer is
limited and the use of external cooling is not possible. Nevertheless, both techniques
have a high efficacy and the choice between the two types of transfer is a matter of
preference.
Once transferred to a membrane, proteins are more readily and equally accessible to
various ligands than they were in the gel. The blot (i.e. the immobilizing matrix
containing the transferred proteins) is therefore reacted with different probes, such as
antibody for the identification of the corresponding antigen, lectin for the detection of
glycoproteins, or ligand for the detection of blotted receptor components, as well as for
studies of protein–ligand associations (see ref. 2; and Chapters 54–55 and 106). The
blot is also widely used with various techniques of protein identification, from which
the measurement of protein mass (by mass spectrometry using nitrocellulose or PVDF
membrane), or the determination of the protein sequence (amino-[N]-terminal Edman
degradation, carboxy-[C]-terminal sequence or amino acid analysis). Recently, a novel
approach has been described for the sensitive analysis and identification of proteins,
separated by 2-D PAGE, transferred onto a PVDF membrane, incubated with succinic acid
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
321