Hoefer TE42 - Manual (Page 25)

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Electrotransfer notes
Electrophoretic transfer advantages
Electrophoretic transfer of proteins and nucleic
acids is much faster than the blotting methods
first described by Southern for DNA, Alwine
et al. for RNA, or Renart et al. for proteins.
The tank transfer method uses high current
to reduce the transfer time of most samples to
45–60 minutes.
Electrophoretic transfer can improve transfer
efficiency over non-electrophoretic blotting,
especially for proteins, but no quantitative
transfer technique has yet been developed due
to the complexity of the reactions. Quantita-
tive recovery is actually not required for most
purposes because binding macromolecules to a
membrane increases the sensitivity of detection
methods such as autoradiography and permits
detection of specific proteins by antibodies or
affinity labels, and of specific nucleic acids by
hybridization with complementary strands of
RNA or DNA.
The buffer can be chosen to result in a transfer
toward either the cathode or the anode. The
buffer pH must be such that all species of interest
are charged and migrate in the same direction.
The ionic strength should not be too high, since
this will produce excessive current and heat.
For this reason, the high salt conditions used by
Southern for capillary blotting of DNA cannot
be used. The most widely used buffer systems are
those of Towbin et al. for transferring proteins,
and of Bittner et al. for transferring nucleic acids.
Buffer systems for transfer of each type of sample
are listed later in this section.