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The blot transfer procedure. In a Southern, or DNA blot transfer, DNA isolated from a cell line or tissue is digested with one or more restriction enzymes.
This mixture is pipetted into a well in an agarose or polyacrylamide gel and exposed to a direct electrical current. DNA, being negatively charged, migrates
toward the anode; the smaller fragments move the most rapidly. After a suitable time, the DNA within the gel is denatured by exposure to mild alkali and
transferred to nitrocellulose or nylon paper, resulting in an exact replica of the pattern on the gel, using the blotting technique devised by Southern. The
DNA is bound to the paper by exposure to heat or UV, and the paper is then exposed to the labeled cDNA probe, which hybridizes to complementary
strands on the filter. After thorough washing, the paper is exposed to x-ray film or an imaging screen, which is developed to reveal several specific bands
Source: Molecular Genetics, Recombinant DNA, & Genomic Technology, Harper's Illustrated Biochemistry, 30e
corresponding to the DNA fragment(s) that were recognized (hybridized to) the sequences in the cDNA probe. The RNA, or Northern, blot is conceptually
Rodwell
VW, Bender DA,
Botham
KM, Kennelly
PJ, Weil some
P. Harper's
Illustrated
Biochemistry,
30e;transfer,
2015 Available
similar. RNA Citation:
is subjected
to electrophoresis
before
blot transfer.
This requires
different
steps from
those of DNA
primarilyat:
to ensure that the
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RNA remains intact, and is generally somewhat more difficult. In the protein, or Western, blot, proteins are electrophoresed and transferred to special
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2017 McGraw-Hill
Education.
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paper that avidly
binds ©proteins
and then probed
with aAllspecific
antibody or other probe molecule. (Asterisks signify labeled probes, either radioactive or
fluorescent.) In the case of Southwestern blotting (see the text; not shown), a protein blot similar to that shown above under “Western” is exposed to