Bio-Rad Criterion™ Blotter - Manual (Page 14)

5. Alter membrane type. As mentioned in 4e, PVDF membrane allows transfer in reduced
alcohol(see Section 5.1). PVDF can increase the binding of low molecular weight
proteins that sometimes blow through nitrocellulose when transfers are long enough or
intense enough to transfer high molecular weight proteins. Use Immun-Blot PVDF if the
blot will be developed with immunochemicals. Use Sequi-Blot PVDF for proteins that will
be sequenced or delivered to mass spec.
6. Increase pore size or decrease %T so that proteins will not be trapped inside small pores.
4.2 Optimizing DNA and RNA Transfer
Problems with elution of nucleic acids can be solved by altering the gel percentage. It
may be somewhat more difficult to quantitatively transfer large amounts of DNA used in
genomic blots. The following tactics should be considered for optimizing elution in such
transfers.
1. Alter gel composition.
a. Lower % total monomer or % crosslinker for polyacrylamide gels.
b. Lower % agarose. This allows better elution of high molecular weight DNA.
2. Alter DNA denaturants. It has been found that glyoxal denaturation allows more efficient
elution of DNA than NaOH. Boiling polyacrylamide gels to denature DNA has also been
found to give excellent results.
11 Base denaturation often causes polyacrylamide gels to
weaken and stick to blotting membranes.
Section 5
Choice of Blotting Membranes
5.1 Protein Blotting Membranes
PVDF Membrane
Bio-Rad offers PVDF (Polyvinylidene difluoride) membranes ideal for immunoassays of
blotted proteins (Immun-Blot PVDF) or amino-terminal sequencing and amino acid analysis
(Sequi-Blot PVDF). PVDF retains proteins under extreme conditions such as exposure to acid,
base, and in the presence of organic solvents. Greater protein binding capacity allows for
better retention of easily transferred proteins, while allowing more time or higher
voltages to transfer difficult or larger proteins. 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.
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. 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 washes, thus limiting detection sensitivity.
19 Smaller pore size nitrocellulose
membrane (0.2 µm), has been shown to be effective in eliminating this loss. 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
12