Bio-Rad Trans-Blot® Cell - Manual (Page 17)

Section 5
Choice of Blotting Membranes
5.1 Protein Blotting Membranes
Nitrocellulose Membrane
Nitrocellulose membranes have been used extensively for protein binding and
detection. 7,20,23,24,27 They can be easily stained for total protein by a dye stain (Amido Black,
Coomassie ® Blue, Ponceau S, Fast Green FCF, etc.), 27 or the more sensitive Colloidal Gold
Total Protein Stain, and also allow either RIA, FIA or EIA. 7 Nitrocellulose has a high bind-
ing capacity of 80-100 µg/cm 2 . Nonspecific protein binding sites are easily and rapidly
blocked, avoiding subsequent background problems. No pre-activation is required. Low
molecular weight proteins (especially <20,000 daltons) may be lost during post transfer wash-
es, thus limiting detection sensitivity.
19 Smaller pore size nitrocellulose membrane (0.2 µm),
has been shown to be effective in eliminating this loss. 37 Large proteins ( 100,000 daltons)
denatured by SDS may transfer poorly due to the addition of alcohol to the transfer buffer.
Alcohol increases binding of SDS-proteins to nitrocellulose, but decreases 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.
17 Also, SDS increases the con-
ductivity of the buffer and the heat generated during transfer.
PVDF Membrane
PVDF (Polyvinylidene difluoride) membrane is an ideal support for amino-terminal
sequencing, amino acid analysis and immunoassays of blotted proteins. PVDF retains proteins
under extreme conditions of exposure to acidic or basic conditions, and in the presence of
organic solvents. Greater retention during sequencing manipulations enhances the likelihood
of obtaining information from rare, low abundance proteins, by increased initial coupling and
higher repetitive yields. In addition, PVDF membrane exhibits better binding efficiency of
blotted material in the presence of SDS in the transfer buffer. PVDF must first be wetted in
100% MeOH but can then be used in buffer which does not contain MeOH.
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 condi-
tions 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.
13 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.
14