Bio-Rad Mini Trans-Blot® Cell - Manual (Page 25)

Mini-Trans-Blot Electrophoretic Transfer Cell 19
Section 5
Choice of Blotting Membranes
5.1 Protein Blotting Membranes
Nitrocellulose Membrane
Nitrocellulose membranes have been used extensively
for protein binding and detection.
8, 21, 24, 25, 28 They can be
easily stained for total protein by a dye stain (Amido
Black, Coomassie Blue, Ponceau S, Fast Green FCF,
etc.),
28 or the more sensitive Colloidal Gold Total Protein
Stain, and also allow either RIA, FIA, or EIA. 8 Nitrocellulose
has a high binding 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 <15,000 daltons) may be lost during
post transfer washes, thus limiting detection sensitivity.
20
Smaller pore size nitrocellulose membrane (0.2 μm),
has been shown to be effective in eliminating this loss.
30
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.
18 Also, SDS increases the conductivity of the
buffer and the heat generated during transfer.
PVDF Membrane
Polyvinylidene difluoride (PVDF) 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.