Bio-Rad Criterion™ Blotter - Manual (Page 15)

pore sizes in the gel. Elimination of alcohol from SDS-protein transfers results in
considerably diminished binding. Adding SDS (up to 0.1%) to the transfer buffer increases the
transfer efficiency of proteins, but reduces the amount of binding to the membrane. Also,
SDS increases the conductivity of the buffer and the heat generated during transfer.
5.2 DNA and RNA Blotting Membrane
Zeta-Probe ® Nylon Membrane
Nitrocellulose is not a suitable medium for electrophoretic transfer of nucleic acids, as high
concentrations of salt (= 10 x SSC) are required for efficient binding. 13 Molecules = 500 bp
are not bound at all, even at high salt. Low resistance results when an electric current is passed
through a solution of high salt. This causes potentially damaging high currents (and power)
at very low voltages. Since V/cm is the eluting force, inefficient transfer occurs under
conditions required for proper binding. Zeta-Probe membrane allows efficient binding of all
sizes of single stranded DNA and RNA in the presence of low ionic strength buffers.
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Zeta-Probe membrane is an ideal alternative to nitrocellulose for the analysis of nucleic acids.
Binding is more stable through post transfer washes, and reprobing may be performed as
many as 10 times.
Table 5.1 Guide to Protein Blotting Membranes
A variety of blotting membranes is available for immunoblotting, each with particular
advantages depending on the needs of the experiment. The physical properties and
performance characteristics of a membrane should be evaluated in selecting the appropriate
transfer conditions.
Binding
Capacity
Membrane Pore Size (µg/cm2) Notes
Nitrocellulose 0.45 µm 80–100 General purpose protein blotting
0.2 µm membrane
Supported 0.45 µm 80–100 Pure nitrocellulose cast on an
Nitrocellulose 0.2 µm inert synthetic support;
Nitrocellulose 0.2 µm increased
strength for easier handling and
for reprobing.
Immun-Blot PVDF 0.2 µm 150–160 High mechanical strength and
chemical stability, used for
immune detection western
blotting; low background to
signal ratio, enhanced binding in
the presence of SDS. Must be
wet in 100% MeOH before
equilibration in buffer.
Sequi-Blot PVDF 0.2 µm 170–200 High mechanical strength and
chemical stability, used for
protein sequencing, enhanced
binding in the presence of SDS.
Must be wet in 100% MeOH
before equilibration in buffer.
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