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

4.2 Optimizing DNA and RNA Transfer
Altering the gel percentage can solve problems with elution of nucleic acids. 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 the 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 the 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
7 . Base denaturation often causes polyacrylamide gels
to weaken and stick to blotting membranes.
Section 5
Choice of Blotting Membranes
5.1 Protein Blotting Membranes
A variety of blotting membranes are available, 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.
Table 5.1 Guide to Protein Blotting Membranes
Membrane Pore Size Binding Notes
Capacity
(
µg/cm 2 )
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; increased
strength for easier handling and for
reprobing.
PVDF 0.2 µm 170–200 High mechanical strength and
chemical stability, used for protein
sequencing and western blotting;
low background to signal ration,
enhanced binding in the presence
of SDS. Must be wet in alcohol
before equilibration in buffer.
Nylon 0.2 µm 170 Recommended for nucleic acids
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