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Technical Advance |
From the Neurobiology Department, University Medical School, Framlington Place, Newcastle upon Tyne, United Kingdom
A multiplex system of Western blotting is presented in which most
of the current muscular dystrophy proteins can be analyzed
simultaneously on one pair of blots. This represents a significant
improvement in efficiency and cost for this type of analysis. The final
diagnosis is more quickly achieved in patients where several possible
diagnoses are indicated after clinical appraisal, and those
with unusual presentations may be quickly resolved. The method uses a
biphasic polyacrylamide gel system, which enables the
corresponding blot to be probed simultaneously with a cocktail of
monoclonal antibodies. The gel is optimized so that large proteins of
more than 200 kd (eg, dystrophin, dysferlin,
and myosin heavy chain) can be analyzed in the top part, while
smaller proteins under 150 kd (eg, calpain 3, the 80-kd
fragment of laminin 
2 chain, all of the
sarcoglycans, and caveolin 3) are separated in the lower phase.
This basic system could be used for different combinations of
antibodies as new muscular dystrophy proteins are identified and
require examination. In addition, analysis of the laminin 2
chain of merosin showed that this protein was expressed as a doublet or
triplet set of bands in many patients with active muscle pathology.
This may indicate the existence of an embryonic isoform, which
is re-expressed in regenerating fibers.
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