Hoefer TE22 - Manual (Page 22)

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Factors affecting the transfer
Parameters such as sample characteristics,
membrane type, gel pore size, and the transfer
buffer used all contribute to the transferability
of macromolecules, and should be kept in mind
when developing a protocol. Very small molecu-
lar species, for instance, migrate quickly but
often do not bind as well as larger molecules;
large molecules bind more efficiently but do not
elute from the gel as rapidly. The rate of elution
is also affected by the pore size of the gel and
the orientation of the molecules.
Further, the degree to which molecules bind
to the membrane is influenced by membrane
characteristics such as pore size and type, and
buffer characteristics such as pH, salt type and
concentration, and the presence of detergents
such as sodium dodecyl sulfate (SDS). Condi-
tions required for efficient elution may not
coincide with optimal conditions for binding.
To find the optimum conditions for transferring
your sample, balance these effects: If the sample
elution rate is slow, a longer transfer period may
be required. (In our experience, low voltage
transfers for longer periods do not offer much
improvement.) If sample binding is inadequate,
try different buffer conditions. For a comprehen-
sive review, see Gershoni and Palade (1983).
If the transfer buffer system is different from the
electrophoresis buffer system, the gel should be
equilibrated with the transfer buffer before the
transfer to ensure swelling or shrinking occurs
before the gel contacts the transfer membrane.
If this step is skipped, band distortion or loss of
resolution could result.