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However, systematic and comprehensive research into the full range of PrP C proteoforms has been hampered by a lack of instruments capable of identifying all PrP C proteoforms. In a complex biological sample, we developed an optimized western blot assay able to obtain the most accurate information about PrP C Constitutive processing and the relative abundance of PrP C proteoforms. Interestingly, the relative amount of shed PrP C in wt mice was higher in wt mice than in most other models. Our results show that constitutive endoproteolytic processing of PrP C is not affected by PrP C overexpression or host factors other than PrP C, although PrP C can be affected by PrP C primary structure.
Source link: https://europepmc.org/article/MED/36565989
Duchenne muscular dystrophy is a degenerative muscular disease that affects approximately one in 5,000 males at birth. New advances in gene therapy and the detection of dystrophin expression in other muscular dystrophies have opened new avenues for therapy. Therefore, accurate techniques are required to monitor dystrophin expression and determine the efficacy of new therapies for muscular dystrophies such as DMD and Becker muscular dystrophy. We'll discuss in most laboratory settings how to validate a novel Western blot technique for the quantitation of mini-dystrophin protein in human skeletal muscle tissue. Workflow for our quantitative WB method to determine mini-dystrophin levels in muscle tissue. Step 1 involves protein extraction from skeletal muscle tissue lysates obtained from control, DMD, or BMD biospecimens. With housekeeping GAPDH, Step 3 involves running gel electrophoresis with wild-type dystrophin from muscle tissue extracts, as well as mini-dystrophin STD curve and mini-DYS and protein normalization.
Source link: https://europepmc.org/article/MED/36539515
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